Human monoclonal antibodies to epidermal growth factor receptor (egfr)

ABSTRACT

Isolated human monoclonal antibodies which specifically bind to human EGFR, and related antibody-based compositions and molecules, are disclosed. The human antibodies can be produced by a transfectoma or in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, non-human transgenic animals and hybridomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/464,057, filed on May 11, 2009, which is a divisional of U.S. patentapplication Ser. No. 10/320,094, filed on Dec. 16, 2002, issued on Sep.29, 2009 as U.S. Pat. No. 7,595,378, which is a continuation-in-part ofU.S. patent application Ser. No. 10/172,317, filed on Jun. 13, 2002,issued on Jul. 24, 2007 as U.S. Pat. No. 7,247,301, which claimspriority to U.S. Provisional Patent Application No. 60/298,172, filed onJun. 13, 2001, the contents of which are incorporated herein in theirentirety by this reference.

BACKGROUND OF THE INVENTION

The EGF receptor (EGFR) is a 170 kDa type 1 transmembrane molecule. Itsexpression is found to be upregulated in many human tumors includingcarcinoma of the head and neck, breast, colon, prostate, lung, andovaries. The degree of over-expression is correlated to poor clinicalprognosis (Baselga, et al. (1994) Pharmac. Therapeut. 64:127-154;Modjtahedi, et al. (1994) Int. J. Oncology 4:277-296). Furthermore, itsexpression is frequently accompanied by the production of EGFR-ligands,TGF-α and EGF among others, by EGFR-expressing tumor cells whichsuggests that an autocrine loop participates in the progression of thesecells (Baselga, et al. (1994) Pharmac. Therapeut. 64:127-154;Modjtahedi, et al. (1994) Int. J. Oncology. 4:277-296). Blocking theinteraction between such EGFR ligands and EGFR therefore can inhibittumor growth and survival (Baselga, et al. (1994) Pharmac. Therapeut.64:127-154).

Monoclonal antibodies (MAbs) directed to the ligand-binding domain ofEGFR can block the interaction with EGF and TGF-α and, concomitantly,the resultant intracellular signaling pathway. Several murine monoclonalantibodies have been generated which achieve such a block in vitro andwhich have been evaluated for their ability to affect tumor growth inmouse xenograft models (Masui, et al. (1986) Cancer Res. 46: 5592-5598;Masui, et al. (1984) Cancer Res. 44: 1002-1007; Goldstein, et al. (1995)Clin. Cancer Res. 1: 1311-1318). When administered one day after thehuman tumor cells, most of the anti-EGFR MAbs were efficacious inpreventing tumor formation in athymic mice (Baselga, et al. (1994)Pharmac. Therapeut. 64:127-154). However, when injected into micebearing established human tumor xenografts, these murine MAbs (e.g.,MAbs 225s and 528) caused only partial tumor regression.Co-administration of chemotherapeutic agents was needed to fullyeradicate the tumors (Baselga, et al. (1994) Pharmac. Therapeut.64:127-154; Fan, et al. (1993) Cancer Res. 53: 4322-4328; Baselga, etal. (1993) J. Natl. Cancer Inst. 85: 1327-1333).

Therefore, while the results obtained to date clearly establish EGFR asa target for immunotherapy, they also show that murine antibodies do notconstitute ideal therapeutic agents. Moreover, treatment with murineantibodies generally triggers severe immune reactions in patients. Tocircumvent the immunogenicity of mouse antibodies, therapeutics shouldideally be fully human. As a step towards this goal, a chimeric versionof the 225 MAb (C225), in which the mouse antibody variable regions arelinked to human constant regions, has been developed. While C225exhibited an improved anti-tumor activity in the treatment ofestablished xenograft tumors in vivo, this was only achieved at highdoses (Goldstein, et al. (1995) Clin. Cancer Res. 1:1311-1318).Currently C225 is being evaluated in clinical trials for treatment ofvarious types of solid tumors (Baselga, J. (2000) J. Clin. Oncol. 18:54S-59S; Baselga, J. (2000) Ann. Oncol. 11 Suppl 3: 187-190, 2000).

Accordingly, the need exists for improved therapeutic antibodies againstEGFR which are effective at treating and/or preventing diseases relatedto overexpression of EGFR when administered at low dosages, and which donot elicit immune reactions in patients. As described above, monoclonalantibodies (MAb) play a prominent role in many diagnostic andtherapeutic approaches to diseases and have become even more attractiveagents with the recent advent of technologies that allow development offully human antibodies. Antibodies and antibody derivatives constitutetwenty five percent of biological drugs currently under development andmany of these are being developed as cancer therapeutics. Antibodiescombine target specificity with the capacity to effectively engage theimmune system. The combination of these properties and their longbiological half-life alerted researchers to the therapeutic potential ofantibodies. This has recently culminated in the U.S. Food and DrugAdministration (FDA) approval of several antibodies for cancertreatment.

SUMMARY OF THE INVENTION

The present invention provides improved antibody therapeutics fortreating and preventing diseases related to expression of EGFR,particularly EGFR-expressing tumors and autoimmune diseases. Theantibodies are improved in that they are fully human (referred to hereinas “HuMAbs™”) and, thus, are less immunogenic in patients. Theantibodies are also therapeutically effective (e.g., at preventinggrowth and/or function of EGFR-expressing cells) at lower dosages thanpreviously reported for other anti-EGFR antibodies. In addition, incertain embodiments, the antibodies have the added benefit of notactivating complement (e.g., not inducing complement mediated lysis oftarget cells) which reduces adverse side-effects during treatment.

Accordingly, in one embodiment, the present invention provides isolatedhuman monoclonal antibodies which specifically bind to human epidermalgrowth factor receptor (EGFR), as well as compositions containing one ora combination of such antibodies. The human antibodies inhibit (e.g.,block) binding of EGFR ligands, such as EGF and TGF-α, to EGFR. Forexample, binding of EGFR ligand to EGFR can be inhibited by at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% andpreferably results in the prevention of EGFR-mediated cell signaling.

Preferred human antibodies of the invention inhibit the growth and/ormediate the killing (e.g., lysis or phagocytosis) of cells expressingEGFR (in vitro or in vivo) in the presence of human effector cells(e.g., polymorphonuclear cells, monocytes, macrophages and dendriticcells), yet they do not activate complement mediated lysis of cellswhich express EGFR. Accordingly, human monoclonal antibodies of theinvention can be used as diagnostic or therapeutic agents in vivo and invitro.

In one embodiment exemplified herein, human antibodies of the inventionare IgG1 (e.g., IgG1k) antibodies having an IgG1 heavy chain and a kappalight chain. However, other antibody isotypes are also encompassed bythe invention, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD,and IgE. The antibodies can be whole antibodies or antigen-bindingfragments of the antibodies, including Fab, F(ab′)₂, Fv and chain Fvfragments.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is an IgG1,κ orIgG1,λ isotype.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is an IgG4antibody.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is an IgG4,κ orIgG4,λ isotype.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises a variable heavy chainamino acid sequence as set forth in SEQ ID NO:2.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises a variable heavy chainamino acid sequence which is at least 90% homologous, preferably atleast 95% homologous, and more preferably at least 98%, or at least 99%homologous to the amino acid sequence as set forth in SEQ ID NO:2.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises a variable light chainamino acid sequence as set forth in SEQ ID NO:4.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises a variable light chainamino acid sequence which is at least 90% homologous, preferably atleast 95% homologous, and more preferably at least 98%, or at least 99%homologous to the amino acid sequence as set forth in SEQ ID NO:4.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises human heavy chain andhuman kappa light chain variable regions which are at least 90%homologous, preferably at least 95% homologous, and more preferably atleast 98%, or at least 99% homologous to the amino acid sequences as setforth in SEQ ID NO:2 and SEQ ID NO:4, respectively.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises at least one CDR sequenceselected from the group consisting of:

(i) the CDR1, CDR2, and CDR3 regions shown in FIG. 15 (SEQ ID NOs:5, 6,7, 8, 9, and 10);

(ii) sequences which are at least 90% homologous, preferably at least95% homologous, and more preferably at least 98%, or at least 99%homologous to the sequences defined in (i); and

(iii) fragments of any one of the sequences defined in (i) or (ii),which retain the ability to bind to human EGFR.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises the heavy chain CDR3region shown in FIG. 15 (SEQ ID NO:7), a sequence which is at least 90%homologous, preferably at least 95% homologous, and more preferably atleast 98%, or at least 99% homologous to SEQ ID NO:7, or a fragmentthereof, which retains the ability to bind to human EGFR.

In another aspect the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises at least four CDRsselected from (i) the CDR regions shown in FIG. 15 (SEQ ID NOs:5, 6, 7,8, 9, or 10); (ii) sequences which are at least 90% homologous,preferably at least 95% homologous, and more preferably at least 98%, orat least 99% homologous to the sequences defined in (i); and (iii)fragments of the sequences defined in (i) or (ii), which retain theability to bind to human EGFR.

In another aspect the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises (i) the 6 CDR regionsshown in FIG. 15 (SEQ ID NOs:5, 6, 7, 8, 9, and 10); (ii) sequenceswhich are at least 90% homologous, preferably at least 95% homologous,and more preferably at least 98%, or at least 99% homologous to thesequences defined in (i); or (iii) fragments of the sequences defined in(i) or (ii), which retain the ability to bind to human EGFR.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is an intactantibody selected from the group consisting of an intact IgG1 antibody,an intact IgG4 antibody, an intact IgM antibody, an intact IgA1antibody, an intact IgA2 antibody, an intact secretory IgA antibody, anintact IgD antibody, and an intact IgE antibody, wherein the antibody isglycosylated in a eukaryotic cell.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is an intactantibody selected from the group consisting of an intact IgG1,κantibody, an intact IgG1,λ antibody, an intact IgG4,κ antibody, and anintact IgG4,κ antibody, wherein the antibody is glycosylated in aeukaryotic cell.

In another aspect, the invention relates to an isolated human monoclonalantibody which binds to human EGFR, wherein the antibody is selectedfrom the group consisting of IgG1, IgA, IgE, IgM, IgG4, and IgDantibodies, and wherein the antibody comprises a heavy chain variableregion amino acid sequence derived from a human V_(H)3-33 germlinesequence (SEQ ID NO:12) and a light chain variable region amino acidsequence derived from a human VκL18 germline sequence (SEQ ID NO:11).

In a particular embodiment, the human antibody is encoded by human IgGheavy chain and human kappa light chain nucleic acids comprisingnucleotide sequences in their variable regions as set forth in SEQ IDNO:1 and SEQ ID NO:3, respectively, and conservative sequencemodifications thereof. In another embodiment, the human antibody includeIgG heavy chain and kappa light chain variable regions which comprisethe amino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4,respectively, and conservative sequence modifications thereof.

In another particular embodiment, the human antibody corresponds toantibody 2F8 or an antibody that binds to the same epitope as (e.g.,competes with) or has the same functional binding characteristics asantibody 2F8.

Human antibodies of the invention can be produced recombinantly in ahost cell, such as a transfectoma (e.g., a transfectoma consisting ofimmortalized CHO cells or lymphocytic cells) containing nucleic acidsencoding the heavy and light chains of the antibody, or be obtaineddirectly from a hybridoma which expresses the antibody (e.g., whichincludes a B cell obtained from a transgenic non-human animal, e.g., atransgenic mouse, having a genome comprising a human heavy chaintransgene and a human light chain transgene that encode the antibody,fused to an immortalized cell). In a particular embodiment, theantibodies are produced by a hybridoma referred to herein as 2F8 or by ahost cell (e.g., a CHO cell) transfectoma containing human heavy chainand human light chain nucleic acids which comprise nucleotide sequencesin their variable regions as set forth in SEQ ID NOs:1 and 3,respectively, and conservative modifications thereof.

In another embodiment, human anti-EGFR antibodies of the presentinvention can be characterized by one or more of the followingproperties:

a) specificity for the EGFR;

b) a binding affinity to EGFR with an equilibrium association constant(K_(A)) of at least about 10⁷ M⁻¹, preferably about, 10⁸ M⁻¹, 10⁹ M⁻¹,and more preferably, about 10¹⁰ M⁻¹ to 10¹¹ M⁻¹ or higher;

c) a dissociation constant (K_(D)) from EGFR of about 10⁻³ s⁻¹,preferably about 10⁻⁴ s⁻¹, more preferably, 10⁻⁵ s⁻¹, and mostpreferably, 10⁻⁶ s⁻¹;

d) the ability to opsonize a cell expressing EGFR; or

e) the ability to inhibit growth and/or mediate phagocytosis and killingof cells expressing EGFR (e.g., a tumor cell) in the presence of humaneffector cells at a concentration of about 10 μg/ml or less (e.g., invitro).

Examples of EGFR-expressing tumor cells which can be targeted (e.g.,opsonized) by human antibodies of the present invention include, but arenot limited to, bladder, breast, colon, kidney, ovarian, prostate, renalcell, squamous cell, lung (non-small cell), or head and neck tumorcells. Other EGFR-expressing cells include synovial fibroblast cells andkeratinocytes which can be used, for example, as target cells in thetreatment of arthritis and psoriasis, respectively.

In another embodiment, human antibodies of the invention bind to EGFRantigen with an affinity constant of at least about 10⁸ M⁻¹, morepreferably at least about 10⁹ M⁻¹ or 10¹⁰ M⁻¹, and are capable ofinhibiting growth and/or mediating phagocytosis and killing of cellsexpressing EGFR by human effector cells, e.g., polymorphonuclear cells(PMNs), monocytes and macrophages, with an IC₅₀ of about 1×10⁻⁷ M orless, or at a concentration of about 10 μg/ml or less in vitro.

In yet another embodiment, human antibodies of the invention inhibitEGFR-mediated cell signaling. For example, the antibodies can inhibitEGFR ligand (e.g., EGF or TGF-α) induced autophosphorylation of EGFR.The antibodies also can inhibit autocrine EGF or TGF-α induced cellactivation or by inducing lysis (ADCC) of EGFR expressing cells in thepresence of human polymorphonuclear cells. Cells which express EGFRinclude, among others, a bladder cell, a breast cell, a colon cell, akidney cell, an ovarian cell, a prostate cell, a renal cell, a squamouscell, a non-small lung cell, a synovial fibroblast cell, and akeratinocyte.

In another aspect, the present invention provides nucleic acid moleculesencoding the antibodies, or antigen-binding portions, of the invention.Recombinant expression vectors which include nucleic acids encodingantibodies of the invention, and host cells transfected with suchvectors, are also encompassed by the invention, as are methods of makingthe antibodies of the invention by culturing such host cells, e.g., anexpression vector comprising a nucleotide sequence encoding the variableand constant regions of the heavy and light chains of antibody 2F8produced by the hybridoma.

In yet another aspect, the invention provides isolated B-cells from atransgenic non-human animal, e.g., a transgenic mouse, which expresshuman anti-EGFR antibodies of the invention. Preferably, the isolated Bcells are obtained from a transgenic non-human animal, e.g., atransgenic mouse, which has been immunized with a purified or enrichedpreparation of EGFR antigen and/or cells expressing the EGFR.Preferably, the transgenic non-human animal, e.g., a transgenic mouse,has a genome comprising a human heavy chain transgene and a human lightchain transgene encoding all or a portion of an antibody of theinvention. The isolated B-cells are then immortalized to provide asource (e.g., a hybridoma) of human anti-EGFR antibodies.

Accordingly, the present invention also provides a hybridoma capable ofproducing human monoclonal antibodies of the invention that specificallybind to EGFR. In one embodiment, the hybridoma includes a B cellobtained from a transgenic non-human animal, e.g., a transgenic mousehaving a genome comprising a human heavy chain transgene and a humanlight chain transgene encoding all or a portion of an antibody of theinvention, fused to an immortalized cell. Particular hybridomas of theinvention include 2F8.

In another aspect the invention relates to a hybridoma comprising a Bcell obtained from a transgenic nonhuman animal in which V-(D)-J genesegment rearrangements have resulted in the formation of a functionalhuman heavy chain transgene and a functional light chain transgene fusedto an immortalized cell, wherein the hybridoma produces a detectableamount of the monoclonal antibody of the invention as defined in any ofthe claims or embodiments herein.

In another aspect the invention relates to a transfectoma comprisingnucleic acids encoding a human heavy chain and a human light chain,wherein the transfectoma produces a detectable amount of the monoclonalantibody of the invention as defined in any of the claims or embodimentsherein.

In yet another aspect, the invention provides a transgenic non-humananimal, such as a transgenic mouse (also referred to herein as a“HuMAb”), which express human monoclonal antibodies that specificallybind to EGFR. In a particular embodiment, the transgenic non-humananimal is a transgenic mouse having a genome comprising a human heavychain transgene and a human light chain transgene encoding all or aportion of an antibody of the invention. The transgenic non-human animalcan be immunized with a purified or enriched preparation of EGFR antigenand/or cells expressing EGFR. Preferably, the transgenic non-humananimal, e.g., the transgenic mouse, is capable of producing multipleisotypes of human monoclonal antibodies to EGFR (e.g., IgG, IgA and/orIgM) by undergoing V-D-J recombination and isotype switching. Isotypeswitching may occur by, e.g., classical or non-classical isotypeswitching.

In another aspect, the present invention provides methods for producinghuman monoclonal antibodies which specifically react with EGFR. In oneembodiment, the method includes immunizing a transgenic non-humananimal, e.g., a transgenic mouse, having a genome comprising a humanheavy chain transgene and a human light chain transgene encoding all ora portion of an antibody of the invention, with a purified or enrichedpreparation of EGFR antigen and/or cells expressing EGFR. B cells (e.g.,splenic B cells) of the animal are then obtained and fused with myelomacells to form immortal, hybridoma cells that secrete human monoclonalantibodies against EGFR.

In yet another aspect, human anti-EGFR antibodies of the invention arederivatized, linked to or co-expressed with another functional molecule,e.g., another peptide or protein (e.g., an Fab′ fragment). For example,an antibody or antigen-binding portion of the invention can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other molecularentities, such as another antibody (e.g., to produce a bispecific or amultispecific antibody), a cytoxin, a cellular ligand or an antigen.Accordingly, present invention encompasses a large variety of antibodyconjugates, bi- and multispecific molecules, and fusion proteins, all ofwhich bind to EGFR expressing cells and which target other molecules tothe cells, or which bind to EGFR and to other molecules or cells.

In a particular embodiment, the invention includes a bispecific ormultispecific molecule comprising at least one first binding specificityfor EGFR (e.g., a human anti-EGFR antibody or fragment or mimeticthereof), and a second binding specificity for an Fc receptor, e.g.,human FcγRI or a human Fcα receptor, or another antigen on an antigenpresenting cell (APC). Typically, bispecific and multispecific moleculesof the invention comprise at least one antibody, or fragment thereof(e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chain Fv), preferably ahuman antibody or a portion thereof, or a “chimeric” or a “humanized”antibody or a portion thereof (e.g., has a variable region, or at leasta complementarity determining region (CDR), derived from a non-humanantibody (e.g., murine) with the remaining portion(s) being human inorigin).

Accordingly, the present invention includes bispecific and multispecificmolecules that bind to both human EGFR and to an Fc receptor, e.g., ahuman IgG receptor, e.g., an Fc-gamma receptor (FcγR), such as FcγRI(CD64), FcγRII (CD32), and FcγRIII (CD16). Other Fc receptors, such ashuman IgA receptors (e.g., FcαRI), also can be targeted. The Fc receptoris preferably located on the surface of an effector cell, e.g., amonocyte, macrophage or an activated polymorphonuclear cell. In apreferred embodiment, the bispecific and multispecific molecules bind toan Fc receptor at a site which is distinct from the immunoglobulin Fc(e.g., IgG or IgA) binding site of the receptor. Therefore, the bindingof the bispecific and multispecific molecules is not blocked byphysiological levels of immunoglobulins.

In another aspect, the present invention provides a conjugate comprisinga human anti-EGFR antibody of the invention linked to a therapeuticmoiety, e.g., a cytotoxic drug, an enzymatically active toxin, or afragment thereof, a radioisotope, or a small molecule anti-cancer drug.

Alternatively, human antibodies of the invention can be co-administeredwith such therapeutic and cytotoxic agents, but not linked to them. Theycan be coadministered simultaneously with such agents (e.g., in a singlecomposition or separately) or can be administered before or afteradministration of such agents. Such agents can include chemotherapeuticagents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate,carmustine, chlorambucil, and cyclophosphamide hydroxyurea. Humanantibodies of the invention also can be administered in conjunction withradiation therapy.

In another aspect, the present invention provides compositions, e.g.,pharmaceutical and diagnostic compositions/kits, comprising apharmaceutically acceptable carrier and at least one human monoclonalantibody of the invention, or an antigen-binding portion thereof, whichspecifically binds to EGFR. In one embodiment, the composition comprisesa combination of the human antibodies or antigen-binding portionsthereof, preferably each of which binds to a distinct epitope. Forexample, a pharmaceutical composition comprising a human monoclonalantibody that mediates highly effective killing of target cells in thepresence of effector cells can be combined with another human monoclonalantibody that inhibits the growth of cells expressing EGFR. Thus, thecombination provides multiple therapies tailored to provide the maximumtherapeutic benefit. Compositions, e.g., pharmaceutical compositions,comprising a combination of at least one human monoclonal antibody ofthe invention, or antigen-binding portions thereof, and at least onebispecific or multispecific molecule of the invention, are also withinthe scope of the invention.

In another aspect the invention relates to a pharmaceutical compositioncomprising the human antibody of the invention as defined in any of theclaims or embodiments herein and a pharmaceutically acceptable carrier.

In another aspect the pharmaceutical composition is in a form suitablefor injection or infusion.

In another aspect the pharmaceutical composition is a liposomeformulation.

In yet another aspect, the invention provides a method for inhibitingthe proliferation and/or growth of a cell expressing EGFR, and/orinducing killing of a cell expressing EGFR, using human antibodies ofthe invention and related compositions as described above. In oneembodiment, the method comprises contacting a cell expressing EGFReither in vitro or in vivo with one or a combination of human monoclonalantibodies of the invention in the presence of a human effector cell.The method can be employed in culture, e.g., in vitro or ex vivo (e.g.,cultures comprising cells expressing EGFR and effector cells). Forexample, a sample containing cells expressing EGFR and effector cellscan be cultured in vitro, and combined with an antibody of theinvention, or an antigen-binding portion thereof (or a bispecific ormultispecific molecule of the invention). Alternatively, the method canbe performed in a subject, e.g., as part of an in vivo (e.g.,therapeutic or prophylactic) protocol.

For use in in vivo treatment and prevention of diseases related to EGFRexpression (e.g., over-expression), human antibodies of the inventionare administered to patients (e.g., human subjects) at therapeuticallyeffective dosages (e.g., dosages which result in growth inhibition,phagocytosis and/or killing of tumor cells expressing EGFR) using anysuitable route of administration, such as injection and other routes ofadministration known in the art for antibody-based clinical products.

Typical EGFR-related diseases which can be treated and/or preventedusing the human antibodies of the invention include, but are not limitedto, autoimmune diseases and cancers. For example, cancers which can betreated and/or prevented include cancer of the bladder, breast,uterine/cervical, colon, kidney, ovarian, prostate, renal cell,pancreatic, colorectal, stomach, squamous cell, lung (non-small cell),esophageal, head and neck. Autoimmune diseases which can be treatedinclude, for example, psoriasis and inflammatory arthritis, e.g.,rheumatoid arthritis, systemic lupus erythematosus-associated arthritis,psoriatic arthritis, Menetrier's disease, systemic sclerosis, Sjögren'ssyndrome, pulmonary fibrosis, bronchial asthma, myelofibrosis, diabeticnephropathy, chronic allograft rejection, chronic glomerulonephritis,Crohn's disease, ulcerative colitis, hepatic cirrhosis, sclerosingcholangitis, chronic uveitis, or cicatricial pemphigoid.

In another aspect the invention relates to a method of treating orpreventing Alzheimer's disease or other forms of dementia.

In one embodiment, the patient is additionally treated with one or morefurther therapeutic agents and/or physical therapies (e.g., radiationtherapy, hyperthermia, transplantation (e.g., bone marrowtransplantation), surgery, sunlight, or phototherapy), such as one ormore further therapeutic agents and/or physical therapies as disclosedin the following. The patient can also be additionally treated with achemotherapeutic agent, radiation, or an agent that modulates, e.g.,enhances or inhibits, the expression or activity of an Fc receptor,e.g., an Fcα receptor or an Fcγ receptor, such as a cytokine. Typicalcytokines for administration during treatment include granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumornecrosis factor (TNF). Typical therapeutic agents include, among others,anti-neoplastic agents such as doxorubicin (adriamycin), cisplatinbleomycin sulfate, carmustine, chlorambucil, and cyclophosphamidehydroxyurea.

The additional therapeutic agents and/or physical therapies may beadministered either prior to, simultaneously with, or followingadministration of the human antibody.

In another aspect the pharmaceutical composition comprises one or morefurther therapeutic agents, such as one or more agents selected fromchemotherapeutic agents, immunosuppressive agent, anti-inflammatoryagents, and anti-psoriasis agents, and/or physical therapies (e.g.,radiation therapy, hyperthermia, transplantation (e.g., bone marrowtransplantation), surgery, sunlight, or phototherapy).

In another aspect the pharmaceutical composition comprises one or morefurther chemotherapeutic agents selected from the group consisting ofnitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines(e.g., thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine and streptozocin), platinum complexes (e.g., carboplatin andcisplatin), non-classical alkylating agents (e.g., dacarbazine andtemozolamide), folate analogs (e.g., methotrexate), purine analogs(e.g., fludarabine and mercaptopurine), adenosine analogs (e.g.,cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil(alone or in combination with leucovorin) and gemcitabine), substitutedureas (e.g., hydroxyurea), antitumor antibiotics (e.g., bleomycin anddoxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide),microtubule agents (e.g., docetaxel and paclitaxel), camptothecinanalogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase),cytokines (e.g., interleukin-2 and interferon-α), monoclonal antibodies(e.g., trastuzumab and bevacizumab), recombinant toxins and immunotoxins(e.g., recombinant cholera toxin-B and TP-38), cancer gene therapies,and cancer vaccines (e.g., vaccine against telomerase). Other treatmentsmay include hyperthermia, radiation therapy, transplantation andsurgery.

In another aspect the pharmaceutical composition comprises one or morefurther therapeutic agents selected from the group consisting ofimmunosuppressive antibodies (e.g., antibodies against MHC, CD2, CD3,CD4, CD7, CD28, B7, CD40, CD45, IFN-γ TNF-α, IL-4, IL-5, IL-6R, IL-7,IL-8, IL-10, CD11a, CD20, or CD58, or antibodies against their ligands)and other immunomodulatory compounds (e.g., soluble IL-15R or IL-10).

In another aspect the pharmaceutical composition comprises one or morefurther immunosuppressive agents selected from the group consisting ofcyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil,corticosteroids (e.g., prednisone), methotrexate, gold salts,sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,tacrolimus (FK-506), OKT3, and anti-thymocyte globulin.

In another aspect the pharmaceutical composition comprises one or morefurther anti-inflammatory agents selected from the group consisting ofaspirin and other salicylates, steroidal drugs, NSAIDs (nonsteroidalanti-inflammatory drugs) (e.g., ibuprofen, fenoprofen, naproxen,sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone,etodolac, oxaprozin, and indomethacin), Cox-2 inhibitors (e.g.,rofecoxib and celecoxib), and DMARDs (disease modifying antirheumaticdrugs) (e.g., methotrexate, hydroxychloroquine, sulfasalazine,azathioprine, pyrimidine synthesis inhibitors (e.g., leflunomide), IL-1receptor blocking agents (e.g., anakinra), TNF-α blocking agents (e.g.,etanercept, infliximab and adalimumab), anti-IL-6R antibodies, CTLA4Ig,and anti-IL-15 antibodies).

In another aspect the pharmaceutical composition comprises one or morefurther anti-psoriasis agents selected from the group consisting of coaltar, A vitamin, anthralin, calcipotrien, tarazotene, corticosteroids,methotrexate, retinoids (e.g., acitretin), cyclosporine, etanercept,alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus(FK-506), and hydroxyurea.

Other treatments may include exposure to sunlight or phototherapy,including UVB (broad-band and narrow-band ultraviolet B), UVA(ultraviolet A) and PUVA (psoralen methoxalen plus ultraviolet A).

In one embodiment the antibodies of the invention are administered incombination with two or more of the above therapies, such as incombination with methotrexate+phototherapy (PUVA or UVA);methotrexate+acitretin; acitretin+phototherapy (PUVA or UVA);methotrexate+acitretin+phototherapy (PUVA or UVB);hydroxyurea+phototherapy (PUVA or UVB); hydroxyurea+acitretin;cyclosporine+methotrexate; or calcipotrien+phototherapy (UVB).

In another aspect, the invention relates to an immunoconjugatecomprising an antibody according to the invention linked to aradioisotope, cytotoxic agent (e.g., calicheamicin and duocarmycin), acytostatic agent, or a chemotherapeutic drug.

In another aspect the invention relates to an immunoconjugate, whereinthe antibody is linked to a radioisotope (e.g., iodine-131, yttrium-90or indium-111).

In another aspect the invention relates to an immunoconjugate, whereinthe antibody is linked to any one of the chemotherapeutic agents asdefined above.

To increase the therapeutic efficacy of human anti-EGFR antibodies ofthe invention against cancer cells which do not highly express EGFR, theantibodies can be co-administered with an agent which upregulates orotherwise effects expression of EGFR, such as a lymphokine preparationwhich cause upregulated and more homogeneous expression of EGFR on tumorcells. Lymphokine preparations suitable for administration includeinterferon-gamma, tumor necrosis factor, and combinations thereof. Thesecan be administered intravenously. Suitable dosages of lymphokinetypically range from 10,000 to 1,000,000 units/patient.

In another aspect, the invention relates to a pharmaceutical compositioncomprising an expression vector comprising a nucleotide sequenceencoding the variable region of a light chain, heavy chain or both lightand heavy chains of a human antibody which binds EGFR.

In another aspect, the invention relates to a pharmaceutical compositioncomprising an expression vector comprising a nucleotide sequenceencoding the variable region of a light chain, heavy chain or both lightand heavy chains of a human antibody which binds EGFR, and furthercomprising a nucleotide sequence encoding the constant region of a lightchain, heavy chain or both light and heavy chains of a human antibodywhich binds EGFR.

In another aspect, the invention relates to a pharmaceutical compositioncomprising an expression vector comprising a nucleotide sequenceencoding heavy chain and light chain variable regions which comprise theamino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively,and conservative sequence modifications thereof.

In yet another aspect, the present invention provides a method fordetecting in vitro or in vivo the presence of EGFR antigen in a sample,e.g., for diagnosing an EGFR-related disease. In one embodiment, this isachieved by contacting a sample to be tested, optionally along with acontrol sample, with a human monoclonal antibody of the invention (or anantigen-binding portion thereof) under conditions that allow forformation of a complex between the antibody and EGFR. Complex formationis then detected (e.g., using an ELISA). When using a control samplealong with the test sample, complex is detected in both samples and anystatistically significant difference in the formation of complexesbetween the samples is indicative the presence of EGFR antigen in thetest sample.

Other features and advantages of the instant invention be apparent fromthe following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing competitive ELISA with hybridoma supernatantsfrom mouse 20241 versus murine monoclonal anti-EGFR MAbs 225, 528, andAB5.

FIG. 2 is a graph showing competitive ELISA with human antibodysupernatants from mouse 20242 and 20243 versus murine monoclonalanti-EGFR MAbs 225, 528, and AB5.

FIG. 3 is a graph showing competitive ELISA with purified humanantibodies versus murine monoclonal anti-EGFR MAbs 225 and 528.

FIGS. 4A, 4B, 4C, and 4D are graphs showing competitive ELISA withHuMabs (A) 6B3, (B) 5F12, (C) 2F8, and (D) 2A2.

FIGS. 5A and 5B are graphs showing inhibition of EGF-biotin binding toEGFR by anti-EGFR HuMabs and murine MAbs (ELISA format).

FIG. 6 is a graph showing inhibition of EGF-biotin binding to EGFR onA431 cells by anti-EGFR HuMabs and murine MAbs.

FIG. 7 is a graph showing titration of anti-EGFR HuMabs on A431 cells.

FIG. 8 is a graph showing the ability of 2F8 to inhibit the binding ofEGF binding to purified and native EGFR. The effect of 2F8 (diamonds),murine 225 (squares), EGF (triangles) or human IgG1 kappa isotypecontrol (bullets) is measured on the binding of EGF-biotin toimmobilized EGFR. As depicted in FIG. 8, 2F8 is able to inhibitEGF-biotin binding with an IC50 of 17 nM, significantly lower than 225(IC50 of 30 nM).

FIG. 9 is a bar graph showing the ability of 2F8 to inhibit the bindingof EGF and TGF-α to A431 cells. A431 cells are derived from an ovarianepidermoid carcinoma and express in excess of 1×10⁶ EGFR molecules ontheir cell surface. Inhibition of 2F8-binding to A431 cells wasdetermined using flow cytometer analysis. Cells were pre-incubated witheither 5 (open bars) or 50 μg/ml (closed bars) ligand before adding 2F8.Binding of antibody without ligand (PBS group) was designated as 100%.As shown, EGF and TGF-α binding to A431 cells is efficiently blocked by2F8. These results indicate that 2F8 binds close to, or at the samesite, on EGFR as the ligands.

FIGS. 10A and 10B show the effect of monoclonal anti EGFR onautophosphorylation of A431 cells. Serum deprived subconfluent A431cells were treated with different antibodies (10 μg/ml) as indicated inthe methods, stimulated with either EGF (A) or TGF-α (B), and extracted.The EGFR phosphorylation was analyzed by SDS-PAGE and immunoblottingwith antiphosphotyrosine antibodies.

FIGS. 11A, 11B, and 11C are graphs showing growth inhibition ofEGFR-expressing tumor cell lines by anti-EGFR human antibodies. TheEGFR-expressing tumor cell lines A431 (A), HN5 (B) and MDA-MB-468 (C)were incubated with various concentrations of HuMab 2F8 (squares), 5C5(triangles), 6E9 (crosses), 2A2 (diamonds) antibody negative controlanti-CTLA4 (open circles), antibody positive control 225 (closedcircles) or with medium only (control) for seven (7) days. Thereafter,cell growth was evaluated using crystal violet staining of fixed cells.The percentage growth inhibition was calculated as the amount of proteinleft after seven (7) days incubation compared to the amount of proteinpresent in the medium only control. The data represent triplicatemeasurements, and are representative of three experiments performed ondifferent days.

FIG. 12 is a graph showing human PMN mediated antibody dependentcellular cytotoxicity. PMN were isolated as described. ⁵¹Chromiumlabeled A431 cells were plated in 96 wells flat bottom plates. PMN wereadded in effector:target ratio 100:1 and antibodies were added indifferent concentrations. After overnight incubation, the ⁵¹Cr releasewas measured.

FIG. 13 is a graph showing the prevention of tumor formation by HuMab2F8 in an athymic murine model. Groups of six (6) mice were injectedsubcutaneously in the flank with 3×10⁶ tumor cells in 200 μl PBS at dayzero (0). Subsequently, mice were injected i.p. on days 1 (75 μg/200μl), 3 (25 μg/200 μl), and 5 (25 μg/200 μl) (arrows) with either HuMab2F8 (closed squares) i.p. of human IgG1-κ MAb as a control (opencircles). The data are presented as mean tumor volume±SEM, and arerepresentative of 3 individual experiments, yielding similar results.

FIG. 14 is a graph showing the eradication of established A431 tumorxenografts by HuMab 2F8 in comparison to murine anti-EGFR MAb (m225).Mice were injected subcutaneously in the flank with 3×10⁶ tumor cells in200 μl PBS on day zero (0). At day 10, mice were randomly allocated totreatment groups and treated on days 12 (75 μg/200 μl), 14 (25 μg/200μl), and 16 (25 μg/200 μl) (arrows) with HuMab 2F8 (closed squares, 2F8short-term) or with murine anti-EGFR MAb 225 (closed triangles, m225short-term). Furthermore, groups were included receiving 75 μg/200 μlHuMab 2F8 or m225 on day 12, continued by 25 μg/200 μl HuMab 2F8 or m225on days 14, 16, 19, 22, 26, 29, 33, 36, and 40 (open squares, 2F8long-term; open triangles, m225 long-term). The data are presented asmean tumor volume+SEM, and are representative of 3 individualexperiments, yielding similar results. Black arrows indicate treatmentdays for the short-term treatment, open arrows indicate treatment daysfor the long-term treatment.

FIGS. 15A and 15B show the nucleotide (SEQ ID NOs:1 and 3) and aminoacid (SEQ ID NOs:2, and 4) sequences of the V_(H)- and V_(L)-regions of2F8, respectively, with CDR regions designated (SEQ ID NOs:5, 6, 7, 8,9, and 10).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel antibody-based therapies fortreating and diagnosing diseases characterized by expression,particularly over-expression, of epidermal growth factor receptorantigen (referred to herein as “EGFR”). Therapies of the inventionemploy isolated human IgG monoclonal antibodies, or antigen-bindingportions thereof, which bind to an epitope present on EGFR. Otherisolated human monoclonal antibodies encompassed by the presentinvention include IgA, IgG1-4, IgE, IgM, and IgD antibodies, e.g.,IgG1,κ or IgG1,λ isotypes, or IgG4,κ or IgG4,λ isotypes. In oneembodiment, the human antibodies are produced in a non-human transgenicanimal, e.g., a transgenic mouse, capable of producing multiple isotypesof human monoclonal antibodies to EGFR (e.g., IgG, IgA and/or IgE) byundergoing V-D-J recombination and isotype switching. Accordingly,aspects of the invention include not only antibodies, antibodyfragments, and pharmaceutical compositions thereof, but also non-humantransgenic animals, B-cells, host cell transfectomas, and hybridomaswhich produce monoclonal antibodies. Methods of using the antibodies ofthe invention to detect a cell expressing EGFR or a related,cross-reactive growth factor receptor, or to inhibit growth,differentiation and/or motility of a cell expressing EGFR, either invitro or in vivo, are also encompassed by the invention.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “epidermal growth factor receptor,” “EGFR,” and “EGFR antigen”are used interchangeably herein, and include variants, isoforms andspecies homologs of human EGFR. In a preferred embodiment, binding of anantibody of the invention to the EGFR-antigen inhibits the growth ofcells expressing EGFR (e.g., a tumor cell) by inhibiting or blockingbinding of EGFR ligand to EGFR. The term “EGFR ligand” encompasses all(e.g., physiological) ligands for EGFR, including EGF, TGF-α, heparinbinding EGF (HB-EGF), amphiregulin (AR), and epiregulin (EPI). Inanother preferred embodiment, binding of an antibody of the invention tothe EGFR-antigen mediates effector cell phagocytosis and/or killing ofcells expressing EGFR.

As used herein, the term “inhibits growth” (e.g., referring to cells) isintended to include any measurable decrease in the growth of a cell whencontacted with an anti-EGFR antibody as compared to the growth of thesame cell not in contact with an anti-EGFR antibody, e.g., theinhibition of growth of a cell by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 99%, or 100%.

As used herein, the terms “inhibits binding” and “blocks binding” (e.g.,referring to inhibition/blocking of binding of EGFR ligand to EGFR) areused interchangeably and encompass both partial and completeinhibition/blocking. The inhibition/blocking of EGFR ligand to EGFRpreferably reduces or alters the normal level or type of cell signalingthat occurs when EGFR ligand binds to EGFR without inhibition orblocking. Inhibition and blocking are also intended to include anymeasurable decrease in the binding affinity of EGFR ligand to EGFR whenin contact with an anti-EGFR antibody as compared to the ligand not incontact with an anti-EGFR antibody, e.g., the blocking of EGFR ligandsto EGFR by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,99%, or 100%.

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein as VH)and a heavy chain constant region. The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as VL)and a light chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (Clq) of the classicalcomplement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., EGFR). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) aF(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentconsisting of the VH and CH1 domains; (iv) a Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody, (v) a dAb fragment(Ward et al., (1989) Nature 341:544-546), which consists of a VH domain;and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are screened forutility in the same manner as are intact antibodies.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “bispecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has two differentbinding specificities. For example, the molecule may bind to, orinteract with, (a) a cell surface antigen and (b) an Fc receptor on thesurface of an effector cell. The term “multispecific molecule” or“heterospecific molecule” is intended to include any agent, e.g., aprotein, peptide, or protein or peptide complex, which has more than twodifferent binding specificities. For example, the molecule may bind to,or interact with, (a) a cell surface antigen, (b) an Fc receptor on thesurface of an effector cell, and (c) at least one other component.Accordingly, the invention includes, but is not limited to, bispecific,trispecific, tetraspecific, and other multispecific molecules which aredirected to cell surface antigens, such as EGFR, and to other targets,such as Fc receptors on effector cells.

The term “bispecific antibodies” also includes diabodies. Diabodies arebivalent, bispecific antibodies in which the VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g.,Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak, R. J., et al. (1994) Structure 2:1121-1123).

As used herein, the term “heteroantibodies” refers to two or moreantibodies, antibody binding fragments (e.g., Fab), derivativestherefrom, or antigen binding regions linked together, at least two ofwhich have different specificities. These different specificitiesinclude a binding specificity for an Fc receptor on an effector cell,and a binding specificity for an antigen or epitope on a target cell,e.g., a tumor cell. The term “human antibody”, as used herein, isintended to include antibodies having variable and constant regionsderived from human germline immunoglobulin sequences. The humanantibodies of the invention may include amino acid residues not encodedby human germline immunoglobulin sequences (e.g., mutations introducedby random or site-specific mutagenesis in vitro or by somatic mutationin vivo). However, the term “human antibody”, as used herein, is notintended to include antibodies in which CDR sequences derived from thegermline of another mammalian species, such as a mouse, have beengrafted onto human framework sequences.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as (a) antibodies isolated from ananimal (e.g., a mouse) that is transgenic for human immunoglobulin genesor a hybridoma prepared therefrom (described further in Section I,below), (b) antibodies isolated from a host cell transformed to expressthe antibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable and constant regionsderived from human germline immunoglobulin sequences. In certainembodiments, however, such recombinant human antibodies can be subjectedto in vitro mutagenesis (or, when an animal transgenic for human Igsequences is used, in vivo somatic mutagenesis) and thus the amino acidsequences of the VH and VL regions of the recombinant antibodies aresequences that, while derived from and related to human germline VH andVL sequences, may not naturally exist within the human antibody germlinerepertoire in vivo.

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

As used herein, a “heterohybrid antibody” refers to an antibody having alight and heavy chains of different organismal origins. For example, anantibody having a human heavy chain associated with a murine light chainis a heterohybrid antibody. Examples of heterohybrid antibodies includechimeric and humanized antibodies, discussed supra.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody thatspecifically binds to EGFR is substantially free of antibodies thatspecifically bind antigens other than EGFR). An isolated antibody thatspecifically binds to an epitope, isoform or variant of human EGFR may,however, have cross-reactivity to other related antigens, e.g., fromother species (e.g., EGFR species homologs). Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals. In one embodiment of the invention, a combination of“isolated” monoclonal antibodies having different specificities arecombined in a well defined composition.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with an affinity ofat least about 1×10⁷ M⁻¹, and binds to the predetermined antigen with anaffinity that is at least two-fold greater than its affinity for bindingto a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

As used herein, the term “high affinity” for an IgG antibody refers to abinding affinity of at least about 10⁷M⁻¹, preferably at least about10⁸M⁻¹, more preferably at least about 10⁹M⁻¹, and still more preferablyat least about 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹ or greater, e.g., up to 10¹³M⁻¹ or greater. However, “high affinity” binding can vary for otherantibody isotypes. For example, “high affinity” binding for an IgMisotype refers to a binding affinity of at least about 1×10⁷M⁻¹.

The term “K_(A)”, as used herein, is intended to refer to theassociation constant of a particular antibody-antigen interaction.

The term “K_(D)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “nonswitched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the CH gene encoding the nonswitched isotype is typically the first CHgene immediately downstream from the functionally rearranged VDJ gene.Isotype switching has been classified as classical or non-classicalisotype switching. Classical isotype switching occurs by recombinationevents which involve at least one switch sequence region in thetransgene. Non-classical isotype switching may occur by, for example,homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion). Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.). As thereis no specific site where recombination always occurs, the final genesequence will typically not be predictable from the construct.

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the nonhuman transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the nonhuman transgenic animal than tothe species from which the CH genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete VH or VL domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding antibodies or antibody portions (e.g., VH, VL,CDR3) that bind to EGFR, is intended to refer to a nucleic acid moleculein which the nucleotide sequences encoding the antibody or antibodyportion are free of other nucleotide sequences encoding antibodies orantibody portions that bind antigens other than EGFR, which othersequences may naturally flank the nucleic acid in human genomic DNA. Inone embodiment, the human anti-EGFR antibody, or portion thereof,includes the nucleotide or amino acid sequence of 2F8, as well as heavychain (VH) and light chain (VL) variable regions having the sequencesshown in SEQ ID NOs:1 and 3, and 2 and 4, respectively.

As disclosed and claimed herein, the sequences set forth in SEQ ID NOs:1-12 include “conservative sequence modifications”, i.e., nucleotide andamino acid sequence modifications which do not significantly affect oralter the binding characteristics of the antibody encoded by thenucleotide sequence or containing the amino acid sequence. Suchconservative sequence modifications include nucleotide and amino acidsubstitutions, additions and deletions. Modifications can be introducedinto SEQ ID NOs:1-12 by standard techniques known in the art, such assite-directed mutagenesis and PCR-mediated mutagenesis. Conservativeamino acid substitutions include ones in which the amino acid residue isreplaced with an amino acid residue having a similar side chain.Families of amino acid residues having similar side chains have beendefined in the art. These families include amino acids with basic sidechains (e.g., lysine, arginine, histidine), acidic side chains (e.g.,aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine,tryptophan), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a human anti-EGFR antibodyis preferably replaced with another amino acid residue from the sameside chain family.

Alternatively, in another embodiment, mutations can be introducedrandomly along all or part of a anti-EGFR antibody coding sequence, suchas by saturation mutagenesis, and the resulting modified anti-EGFRantibodies can be screened for binding activity.

Accordingly, antibodies encoded by the (heavy and light chain variableregion) nucleotide sequences disclosed herein and/or containing the(heavy and light chain variable region) amino acid sequences disclosedherein (i.e., SEQ ID NOs:1-12) include substantially similar antibodiesencoded by or containing similar sequences which have beenconservatively modified. Further discussion as to how such substantiallysimilar antibodies can be generated based on the partial (i.e., heavyand light chain variable regions) sequences disclosed herein as SEQ IDNOs:1-12 is provided below.

For nucleic and amino acids, the term “substantial homology” indicatesthat two nucleic/amino acids, or designated sequences thereof, whenoptimally aligned and compared, are identical, with appropriatenucleotide/amino acid residue insertions or deletions, in at least about80% of the nucleotides/amino acid residues, usually at least about 90%to 95%, and more preferably at least about 98% to 99.5% of thenucleotides/amino acid residues. Alternatively, substantial homologyexists for nucleic acids when the segments will hybridize underselective hybridization conditions, to the complement of the strand.

For nucleotide acid and amino acid sequences, the term “homology”indicates the degree of identity between two sequences, when optimallyaligned and compared, with appropriate nucleotide insertions ordeletions.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Thepercent identity between two nucleotide or amino acid sequences can alsodetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures may be mutated, thereof inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butto the progeny of such a cell. Because certain modifications may occurin succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, CHO cells andlymphocytic cells.

The term “transfectoma”, as used herein, includes recombinant eukaryotichost cell expressing the antibody, such as CHO cells or NS/0 cells.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

The terms “transgenic, nonhuman animal” refers to a nonhuman animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. For example, a transgenic mouse can have a humanlight chain transgene and either a human heavy chain transgene or humanheavy chain transchromosome, such that the mouse produces humananti-EGFR antibodies when immunized with EGFR and/or cells expressingEGFR. The human heavy chain transgene can be integrated into thechromosomal DNA of the mouse, as is the case for transgenic, e.g., HuMAbmice, or the human heavy chain transgene can be maintainedextrachromosomally, as is the case for transchromosomal (e.g., KM) miceas described in WO 02/43478. Such transgenic and transchromosomal miceare capable of producing multiple isotypes of human monoclonalantibodies to EGFR (e.g., IgG, IgA and/or IgE) by undergoing V-D-Jrecombination and isotype switching.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Production of Human Antibodies to EGFR

The monoclonal antibodies (MAbs) of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256: 495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

In a preferred embodiment, human monoclonal antibodies directed againstEGFR can be generated using transgenic mice carrying parts of the humanimmune system rather than the mouse system. These transgenic mice,referred to herein as “HuMAb” mice, contain a human immunoglobulin geneminiloci that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, et al. (1994)Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed inLonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101;Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol. 13: 65-93,and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci.764:536-546). The preparation of HuMAb mice is described in detailSection II below and in Taylor, L. et al. (1992) Nucleic Acids Research20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724;Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993)EMBO J. 12: 821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920;Lonberg et al., (1994) Nature 368(6474): 856-859; Lonberg, N. (1994)Handbook of Experimental Pharmacology 113:49-101; Taylor, L. et al.(1994) International Immunology 6: 579-591; Lonberg, N. and Huszar, D.(1995) Intern. Rev. Immunol. Vol. 13: 65-93; Harding, F. and Lonberg, N.(1995) Ann. N.Y. Acad. Sci. 764:536-546; Fishwild, D. et al. (1996)Nature Biotechnology 14: 845-851, the contents of all of which arehereby incorporated by reference in their entirety. See further, U.S.Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all toLonberg and Kay, and GenPharm International; U.S. Pat. No. 5,545,807 toSurani et al.; International Publication Nos. WO 98/24884, published onJun. 11, 1998; WO 94/25585, published Nov. 10, 1994; WO 93/1227,published Jun. 24, 1993; WO 92/22645, published Dec. 23, 1992; WO92/03918, published Mar. 19, 1992, the disclosures of all of which arehereby incorporated by reference in their entity. Alternatively, theHCO12 transgenic mice described in Example 2, can be used to generatehuman anti-EGFR antibodies.

Human Antibody Immunizations

To generate fully human monoclonal antibodies to EGFR, HuMAb mice can beimmunized with a purified or enriched preparation of EGFR antigen and/orcells expressing EGFR, as described by Lonberg, N. et al. (1994) Nature368(6474): 856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851 and WO 98/24884. Preferably, the mice will be 6-16 weeks of ageupon the first infusion. For example, a purified or enriched preparation(5-20 μg) of EGFR antigen (e.g., purified from EGFR-expressing LNCaPcells) can be used to immunize the HuMAb mice intraperitoneally. In theevent that immunizations using a purified or enriched preparation ofEGFR antigen do not result in antibodies, mice can also be immunizedwith cells expressing EGFR, e.g., a tumor cell line, to promote immuneresponses.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) with antigen in complete Freund's adjuvant, followed by every otherweek i.p. immunizations (up to a total of 6) with antigen in incompleteFreund's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA (as describedbelow), and mice with sufficient titers of anti-EGFR humanimmunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen. It is expected that 2-3 fusions for each antigen may need to beperformed. Several mice will be immunized for each antigen. For example,a total of twelve HuMAb mice of the HC07 and HC012 strains can beimmunized.

Generation of Hybridomas Producing Human Monoclonal Antibodies to EGFR

The mouse splenocytes can be isolated and fused with PEG to a mousemyeloma cell line based upon standard protocols. The resultinghybridomas are then screened for the production of antigen-specificantibodies. For example, single cell suspensions of splenic lymphocytesfrom immunized mice are fused to one-sixth the number of P3X63-Ag8.653nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cellsare plated at approximately 2×10⁵ in flat bottom microtiter plate,followed by a two week incubation in selective medium containing 20%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM L˜glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50mg/ml gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After two weeks, cells are cultured in medium in which the HATis replaced with HT. Individual wells are then screened by ELISA forhuman anti-EGFR monoclonal IgM and IgG antibodies. Once extensivehybridoma growth occurs, medium is observed usually after 10-14 days.The antibody secreting hybridomas are replated, screened again, and ifstill positive for human IgG, anti-EGFR monoclonal antibodies, can besubcloned at least twice by limiting dilution. The stable subclones arethen cultured in vitro to generate small amounts of antibody in tissueculture medium for characterization.

Generation of Transfectomas Producing Human Monoclonal Antibodies toEGFR

Human antibodies of the invention can also be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification, site directed mutagenesis) and can be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr-CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS/0 myeloma cells, COS cells and SP2.0 cells. Inparticular for use with NS/0 myeloma cells, another preferred expressionsystem is the GS gene expression system disclosed in WO 87/04462, WO89/01036 and EP 338 841. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody atindividual evenly across the variable region. For example, somaticmutations are relatively infrequent in the amino-terminal portion offramework region. For example, somatic mutations are relativelyinfrequent in the amino terminal portion of framework region 1 and inthe carboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see PCT/US99/05535 filed on Mar. 12, 1999, which isherein incorporated by referenced for all purposes). Partial heavy andlight chain sequence spanning the CDR regions is typically sufficientfor this purpose. The partial sequence is used to determine whichgermline variable and joining gene segments contributed to therecombined antibody variable genes. The germline sequence is then usedto fill in missing portions of the variable regions. Heavy and lightchain leader sequences are cleaved during protein maturation and do notcontribute to the properties of the final antibody. For this reason, itis necessary to use the corresponding germline leader sequence forexpression constructs. To add missing sequences, cloned cDNA sequencescab be combined with synthetic oligonucleotides by ligation or PCRamplification. Alternatively, the entire variable region can besynthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has certain advantages such as elimination orinclusion or particular restriction sites, or optimization of particularcodons.

The nucleotide sequences of heavy and light chain transcripts from ahybridomas are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak,1991, J. Biol. Chem. 266:19867-19870); and, HindIII sites are engineeredupstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assemble into overlapping double stranded sets that span segmentsof 150-400 nucleotides. The pools are then used as templates to producePCR amplification products of 150-400 nucleotides. Typically, a singlevariable region oligonucleotide set will be broken down into two poolswhich are separately amplified to generate two overlapping PCV products.These overlapping products are then combined by PCT amplification toform the complete variable region. It may also be desirable to includean overlapping fragment of the heavy or light chain constant region(including the BbsI site of the kappa light chain, or the AgeI site ifthe gamma heavy chain) in the PCR amplification to generate fragmentsthat can easily be cloned into the expression vector constructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, translation initiation, constant region,3′ untranslated, polyadenylation, and transcription termination,sequences to form expression vector constructs. The heavy and lightchain expression constructs can be combined into a single vector,co-transfected, serially transfected, or separately transfected intohost cells which are then fused to form a host cell expressing bothchains.

Plasmids for use in construction of expression vectors for human IgGκare described below. The plasmids were constructed so that PCR amplifiedV heavy and V kappa light chain cDNA sequences could be used toreconstruct complete heavy and light chain minigenes. These plasmids canbe used to express completely human, or chimeric IgG₁κ or IgG₄κantibodies. Similar plasmids can be constructed for expression of otherheavy chain isotypes, or for expression of antibodies comprising lambdalight chains.

Thus, in another aspect of the invention, the structural features of anhuman anti-EGFR antibodies of the invention, e.g., 2F8, are used tocreate structurally related human anti-EGFR antibodies that retain atleast one functional property of the antibodies of the invention, suchas binding to EGFR. More specifically, one or more CDR regions of 2F8can be combined recombinantly with known human framework regions andCDRs to create additional, recombinantly-engineered, human anti-EGFRantibodies of the invention.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-EGFR antibody comprising:

preparing an antibody comprising (1) human heavy chain framework regionsand human heavy chain CDRs, wherein at least one of the human heavychain CDRs comprises an amino acid sequence selected from the amino acidsequences of CDRs shown in FIG. 15 (SEQ ID NOs:5, 6, and 7); and (2)human light chain framework regions and human light chain CDRs, whereinat least one of the human light chain CDRs comprises an amino acidsequence selected from the amino acid sequences of CDRs shown in FIG. 15(SEQ ID NOs:8, 9, and 10); wherein the antibody retains the ability tobind to EGFR.

The ability of the antibody to bind EGFR can be determined usingstandard binding assays, such as those set forth in the Examples (e.g.,an ELISA). Since it is well known in the art that antibody heavy andlight chain CDR3 domains play a particularly important role in thebinding specificity/affinity of an antibody for an antigen, therecombinant antibodies of the invention prepared as set forth abovepreferably comprise the heavy and light chain CDR3s of 2F8. Theantibodies further can comprise the CDR2s of 2F8. The antibodies furthercan comprise the CDR1s of 2F8. Accordingly, the invention furtherprovides anti-EGFR antibodies comprising: (1) human heavy chainframework regions, a human heavy chain CDR1 region, a human heavy chainCDR2 region, and a human heavy chain CDR3 region, wherein the humanheavy chain CDR3 region is the CDR3 of 2F8 as shown in FIG. 15 (SEQ IDNO:7); and (2) human light chain framework regions, a human light chainCDR1 region, a human light chain CDR2 region, and a human light chainCDR3 region, wherein the human light chain CDR3 region is the CDR3 of2F8 as shown in FIG. 15 (SEQ ID NO:10), wherein the antibody binds EGFR.The antibody may further comprise the heavy chain CDR2 and/or the lightchain CDR2 of 2F8. The antibody may further comprise the heavy chainCDR1 and/or the light chain CDR1 of 2F8.

Preferably, the CDR1, 2, and/or 3 of the engineered antibodies describedabove comprise the exact amino acid sequence(s) as those of 2F8disclosed herein. However, the ordinarily skilled artisan willappreciate that some deviation from the exact CDR sequences of 2F8 maybe possible while still retaining the ability of the antibody to bindEGFR effectively (e.g., conservative substitutions). Accordingly, inanother embodiment, the engineered antibody may be composed of one ormore CDRs that are, for example, 90%, 95%, 98% or 99.5% identical to oneor more CDRs of 2F8.

In addition or alternative, to simply binding EGFR, engineeredantibodies such as those described above may be selected for theirretention of other functional properties of antibodies of the invention,such as:

(1) binding to live cells expressing EGFR;

(2) high affinity binding to EGFR;

(3) binding to a unique epitope on EGFR (to eliminate the possibilitythat monoclonal antibodies with complimentary activities when used incombination would compete for binding to the same epitope);

(4) opsonization of cells expressing EGFR; and/or

(5) mediation of growth inhibition, phagocytosis and/or killing of cellsexpressing EGFR in the presence of human effector cells.

Characterization of Binding of Human Monoclonal Antibodies to EGFR

To characterize binding of human monoclonal EGFR antibodies of theinvention, sera from immunized mice can be tested, for example, byELISA. Briefly, microtiter plates are coated with purified EGFR at 0.25μg/ml in PBS, and then blocked with 5% bovine serum albumin in PBS.Dilutions of plasma from EGFR-immunized mice are added to each well andincubated for 1-2 hours at 37° C. The plates are washed with PBS/Tweenand then incubated with a goat-anti-human IgG Fc-specific polyclonalreagent conjugated to alkaline phosphatase for 1 hour at 37° C. Afterwashing, the plates are developed with pNPP substrate (1 mg/ml), andanalyzed at OD of 405-650. Preferably, mice which develop the highesttiters will be used for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with EGFR immunogen. Hybridomasthat bind with high avidity to EGFR will be subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify human anti-EGFR antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected human anti-EGFR monoclonal antibodies bindto unique epitopes, each antibody can be biotinylated using commerciallyavailable reagents (Pierce, Rockford, Ill.). Competition studies usingunlabeled monoclonal antibodies and biotinylated monoclonal antibodiescan be performed using EGFR coated-ELISA plates as described above.Biotinylated MAb binding can be detected with a strep-avidin-alkalinephosphatase probe.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed. Wells of microtiter plates can be coated with 10 g/ml ofanti-human Ig overnight at 4° C. After blocking with 5% BSA, the platesare reacted with 10 g/ml of monoclonal antibodies or purified isotypecontrols, at ambient temperature for two hours. The wells can then bereacted with either human IgGl or human IgM-specific alkalinephosphatase-conjugated probes. Plates are developed and analyzed asdescribed above.

In order to demonstrate binding of monoclonal antibodies to live cellsexpressing the EGFR, flow cytometry can be used. Briefly, cell linesexpressing EGFR (grown under standard growth conditions) are mixed withvarious concentrations of monoclonal antibodies in PBS containing 0.1%Tween 80 and 20% mouse serum, and incubated at 37° C. for 1 hour. Afterwashing, the cells are reacted with Fluorescein-labeled anti-human IgGantibody under the same conditions as the primary antibody staining. Thesamples can be analyzed by FACScan instrument using light and sidescatter properties to gate on single cells. An alternative assay usingfluorescence microscopy may be used (in addition to or instead of) theflow cytometry assay. Cells can be stained exactly as described aboveand examined by fluorescence microscopy. This method allowsvisualization of individual cells, but may have diminished sensitivitydepending on the density of the antigen.

Anti-EGFR human IgGs can be further tested for reactivity with EGFRantigen by Western blotting. Briefly, cell extracts from cellsexpressing EGFR can be prepared and subjected to sodium dodecyl sulfate(SDS) polyacrylamide gel electrophoresis. After electrophoresis, theseparated antigens will be transferred to nitrocellulose membranes,blocked with 20% mouse serum, and probed with the monoclonal antibodiesto be tested. Human IgG binding can be detected using anti-human IgGalkaline phosphatase and developed with BCIP/NBT substrate tablets(Sigma Chem. Co., St. Louis, Mo.).

Phagocytic and Cell Killing Activities of Human Monoclonal Antibodies toEgfr

In addition to binding specifically to EGFR, human monoclonal anti-EGFRantibodies can be tested for their ability to mediate phagocytosis andkilling of cells expressing EGFR. The testing of monoclonal antibodyactivity in vitro will provide an initial screening prior to testing invivo models. Briefly, polymorphonuclear cells (PMN), or other effectorcells, from healthy donors can be purified by Ficoll Hypaque densitycentrifugation, followed by lysis of contaminating erythrocytes. WashedPMNs, can be suspended in RPMI supplemented with 10% heat-inactivatedfetal calf serum and mixed with ⁵¹Cr labeled cells expressing EGFR, atvarious ratios of effector cells to tumor cells (-effector cells:tumorcells). Purified human anti-EGFR IgGs can then be added at variousconcentrations. Irrelevant human IgG can be used as negative control.Assays can be carried out for 0-120 minutes at 37° C. Samples can beassayed for cytolysis by measuring ⁵¹Cr release into the culturesupernatant. Anti-EGFR monoclonal can also be tested in combinationswith each other to determine whether cytolysis is enhanced with multiplemonoclonal antibodies.

Human monoclonal antibodies which bind to EGFR also can be tested in anin vivo model (e.g., in mice) to determine their efficacy in mediatingphagocytosis and killing of cells expressing EGFR, e.g., tumor cells.These antibodies can be selected, for example, based on the followingcriteria, which are not intended to be exclusive:

1.) binding to live cells expressing EGFR;

2.) high affinity of binding to EGFR;

3.) binding to a unique epitope on EGFR (to eliminate the possibilitythat monoclonal antibodies with complimentary activities when used incombination would compete for binding to the same epitope);

4.) opsonization of cells expressing EGFR;

5.) mediation of growth inhibition, phagocytosis and/or killing of cellsexpressing EGFR in the presence of human effector cells.

Preferred human monoclonal antibodies of the invention meet one or more,and preferably all, of these criteria. In a particular embodiment, thehuman monoclonal antibodies are used in combination, e.g., as apharmaceutical composition comprising two or more anti-EGFR monoclonalantibodies or fragments thereof. For example, human anti-EGFR monoclonalantibodies having different, but complementary activities can becombined in a single therapy to achieve a desired therapeutic ordiagnostic effect. An illustration of this would be a compositioncontaining an anti-EGFR human monoclonal antibody that mediates highlyeffective killing of target cells in the presence of effector cells,combined with another human anti-EGFR monoclonal antibody that inhibitsthe growth of cells expressing EGFR.

II. Production of Transgenic Nonhuman Animals which Generate HumanMonoclonal Anti-EGFR Antibodies

In yet another aspect, the invention provides transgenic non-humananimals, e.g., a transgenic mice, which are capable of expressing humanmonoclonal antibodies that specifically bind to EGFR, preferably withhigh affinity. In a preferred embodiment, the transgenic non-humananimals, e.g., the transgenic mice (HuMAb mice), have a genomecomprising a human heavy chain transgene and a light chain transgene. Inone embodiment, the transgenic non-human animals, e.g., the transgenicmice, have been immunized with a purified or enriched preparation ofEGFR antigen and/or cells expressing EGFR. Preferably, the transgenicnon-human animals, e.g., the transgenic mice, are capable of producingmultiple isotypes of human monoclonal antibodies to EGFR (e.g., IgG, IgAand/or IgE) by undergoing V-D-J recombination and isotype switching.Isotype switching may occur by, e.g., classical or non-classical isotypeswitching.

The design of a transgenic non-human animal that responds to foreignantigen stimulation with a heterologous antibody repertoire, requiresthat the heterologous immunoglobulin transgenes contain within thetransgenic animal function correctly throughout the pathway of B-celldevelopment. In a preferred embodiment, correct function of aheterologous heavy chain transgene includes isotype switching.Accordingly, the transgenes of the invention are constructed so as toproduce isotype switching and one or more of the following: (1) highlevel and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

Not all of the foregoing criteria need be met. For example, in thoseembodiments wherein the endogenous immunoglobulin loci of the transgenicanimal are functionally disrupted, the transgene need not activateallelic exclusion. Further, in those embodiments wherein the transgenecomprises a functionally rearranged heavy and/or light chainimmunoglobulin gene, the second criteria of functional generearrangement is unnecessary, at least for that transgene which isalready rearranged. For background on molecular immunology, see,Fundamental Immunology, 2nd edition (1989), Paul William E., ed. RavenPress, N.Y., which is incorporated herein by reference.

In certain embodiments, the transgenic non-human animals used togenerate the human monoclonal antibodies of the invention containrearranged, unrearranged or a combination of rearranged and unrearrangedheterologous immunoglobulin heavy and light chain transgenes in thegermline of the transgenic animal. Each of the heavy chain transgenescomprises at least one C_(H) gene. In addition, the heavy chaintransgene may contain functional isotype switch sequences, which arecapable of supporting isotype switching of a heterologous transgeneencoding multiple C_(H) genes in the B-cells of the transgenic animal.Such switch sequences may be those which occur naturally in the germlineimmunoglobulin locus from the species that serves as the source of thetransgene C_(H) genes, or such switch sequences may be derived fromthose which occur in the species that is to receive the transgeneconstruct (the transgenic animal). For example, a human transgeneconstruct that is used to produce a transgenic mouse may produce ahigher frequency of isotype switching events if it incorporates switchsequences similar to those that occur naturally in the mouse heavy chainlocus, as presumably the mouse switch sequences are optimized tofunction with the mouse switch recombinase enzyme system, whereas thehuman switch sequences are not. Switch sequences may be isolated andcloned by conventional cloning methods, or may be synthesized de novofrom overlapping synthetic oligonucleotides designed on the basis ofpublished sequence information relating to immunoglobulin switch regionsequences (Mills et al., Nucl. Acids Res. 15:7305-7316 (1991); Sideraset al., Intl. Immunol. 1:631-642 (1989), which are incorporated hereinby reference). For each of the foregoing transgenic animals,functionally rearranged heterologous heavy and light chainimmunoglobulin transgenes are found in a significant fraction of theB-cells of the transgenic animal (at least 10 percent).

The transgenes used to generate the transgenic animals of the inventioninclude a heavy chain transgene comprising DNA encoding at least onevariable gene segment, one diversity gene segment, one joining genesegment and at least one constant region gene segment. Theimmunoglobulin light chain transgene comprises DNA encoding at least onevariable gene segment, one joining gene segment and at least oneconstant region gene segment. The gene segments encoding the light andheavy chain gene segments are heterologous to the transgenic non-humananimal in that they are derived from, or correspond to, DNA encodingimmunoglobulin heavy and light chain gene segments from a species notconsisting of the transgenic non-human animal. In one aspect of theinvention, the transgene is constructed such that the individual genesegments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes support recombination of the V, D, and J gene segments(functional rearrangement) and preferably support incorporation of allor a portion of a D region gene segment in the resultant rearrangedimmunoglobulin heavy chain within the transgenic non-human animal whenexposed to EGFR antigen.

In an alternate embodiment, the transgenes comprise an unrearranged“mini-locus.” Such transgenes typically comprise a substantial portionof the C, D, and J segments as well as a subset of the V gene segments.In such transgene constructs, the various regulatory sequences, e.g.,promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences may be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments may becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences may be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif. For example, aminilocus comprises a portion of the genomic immunoglobulin locus havingat least one internal (i.e., not at a terminus of the portion) deletionof a non-essential DNA portion (e.g., intervening sequence; intron orportion thereof) as compared to the naturally-occurring germline Iglocus.

In a preferred embodiment of the invention, the transgenic animal usedto generate human antibodies to EGFR contains at least one, typically2-10, and sometimes 25-50 or more copies of the transgene described inExample 12 of WO 98/24884 (e.g., pHC1 or pHC2) bred with an animalcontaining a single copy of a light chain transgene described inExamples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with theJ_(H) deleted animal described in Example 10 of WO 98/24884, thecontents of which are hereby expressly incorporated by reference.Animals are bred to homozygosity for each of these three traits. Suchanimals have the following genotype: a single copy (per haploid set ofchromosomes) of a human heavy chain unrearranged mini-locus (describedin Example 12 of WO 98/24884), a single copy (per haploid set ofchromosomes) of a rearranged human K light chain construct (described inExample 14 of WO 98/24884), and a deletion at each endogenous mouseheavy chain locus that removes all of the functional J_(H) segments(described in Example 10 of WO 98/24884). Such animals are bred withmice that are homozygous for the deletion of the J_(H) segments(Examples 10 of WO 98/24884) to produce offspring that are homozygousfor the J_(H) deletion and hemizygous for the human heavy and lightchain constructs. The resultant animals are injected with antigens andused for production of human monoclonal antibodies against theseantigens.

B cells isolated from such an animal are monospecific with regard to thehuman heavy and light chains because they contain only a single copy ofeach gene. Furthermore, they will be monospecific with regards to humanor mouse heavy chains because both endogenous mouse heavy chain genecopies are nonfunctional by virtue of the deletion spanning the J_(H)region introduced as described in Example 9 and 12 of WO 98/24884.Furthermore, a substantial fraction of the B cells will be monospecificwith regards to the human or mouse light chains because expression ofthe single copy of the rearranged human κ light chain gene willallelically and isotypically exclude the rearrangement of the endogenousmouse κ and lambda chain genes in a significant fraction of B-cells.

The transgenic mouse of the preferred embodiment will exhibitimmunoglobulin production with a significant repertoire, ideallysubstantially similar to that of a native mouse. Thus, for example, inembodiments where the endogenous Ig genes have been inactivated, thetotal immunoglobulin levels will range from about 0.1 to 10 mg/ml ofserum, preferably 0.5 to 5 mg/ml, ideally at least about 1.0 mg/ml. Whena transgene capable of effecting a switch to IgG from IgM has beenintroduced into the transgenic mouse, the adult mouse ratio of serum IgGto IgM is preferably about 10:1. The IgG to IgM ratio will be much lowerin the immature mouse. In general, greater than about 10%, preferably 40to 80% of the spleen and lymph node B cells express exclusively humanIgG protein.

The repertoire will ideally approximate that shown in a non-transgenicmouse, usually at least about 10% as high, preferably 25 to 50% or more.Generally, at least about a thousand different immunoglobulins (ideallyIgG), preferably 10⁴ to 10⁶ or more, will be produced, dependingprimarily on the number of different V, J and D regions introduced intothe mouse genome. These immunoglobulins will typically recognize aboutone-half or more of highly antigenic proteins, e.g., staphylococcusprotein A. Typically, the immunoglobulins will exhibit an affinity forpreselected antigens of at least about 10⁷M⁻¹, preferably at least about10⁹M⁻¹, more preferably at least about 10¹⁰ M⁻¹, 10¹¹ M⁻¹, 10¹² M⁻¹, orgreater, e.g., up to 10¹³M⁻¹ or greater.

In some embodiments, it may be preferable to generate mice withpredetermined repertoires to limit the selection of V genes representedin the antibody response to a predetermined antigen type. A heavy chaintransgene having a predetermined repertoire may comprise, for example,human VH genes which are preferentially used in antibody responses tothe predetermined antigen type in humans. Alternatively, some VH genesmay be excluded from a defined repertoire for various reasons (e.g.,have a low likelihood of encoding high affinity V regions for thepredetermined antigen; have a low propensity to undergo somatic mutationand affinity sharpening; or are immunogenic to certain humans). Thus,prior to rearrangement of a transgene containing various heavy or lightchain gene segments, such gene segments may be readily identified, e.g.,by hybridization or DNA sequencing, as being from a species of organismother than the transgenic animal.

The transgenic mice of the present invention can be immunized with apurified or enriched preparation of EGFR antigen and/or cells expressingEGFR as described previously. The mice will produce B cells whichundergo class-switching via intratransgene switch recombination(cis-switching) and express immunoglobulins reactive with EGFR. Theimmunoglobulins can be human sequence antibodies, wherein the heavy andlight chain polypeptides are encoded by human transgene sequences, whichmay include sequences derived by somatic mutation and V regionrecombinatorial joints, as well as germline-encoded sequences; thesehuman sequence immunoglobulins can be referred to as being substantiallyidentical to a polypeptide sequence encoded by a human V_(L) or V_(H)gene segment and a human J_(L) or J_(L) segment, even though othernon-germline sequences may be present as a result of somatic mutationand differential V-J and V-D-J recombination joints. With respect tosuch human sequence antibodies, the variable regions of each chain aretypically at least 80 percent encoded by human germline V, J, and, inthe case of heavy chains, D, gene segments; frequently at least 85percent of the variable regions are encoded by human germline sequencespresent on the transgene; often 90 or 95 percent or more of the variableregion sequences are encoded by human germline sequences present on thetransgene. However, since non-germline sequences are introduced bysomatic mutation and VJ and VDJ joining, the human sequence antibodieswill frequently have some variable region sequences (and less frequentlyconstant region sequences) which are not encoded by human V, D, or Jgene segments as found in the human transgene(s) in the germline of themice. Typically, such non-germline sequences (or individual nucleotidepositions) will cluster in or near CDRs, or in regions where somaticmutations are known to cluster.

The human sequence antibodies which bind to the predetermined antigencan result from isotype switching, such that human antibodies comprisinga human sequence γ chain (such as γ1, γ2a, γ2B, or γ3) and a humansequence light chain (such as K) are produced. Such isotype-switchedhuman sequence antibodies often contain one or more somatic mutation(s),typically in the variable region and often in or within about 10residues of a CDR) as a result of affinity maturation and selection of Bcells by antigen, particularly subsequent to secondary (or subsequent)antigen challenge. These high affinity human sequence antibodies mayhave binding affinities of at least 1×10⁹ M⁻¹, typically at least 5×10⁹M⁻¹, frequently more than 1×10¹⁰ M⁻¹, and sometimes 5×10¹⁰ M⁻¹ to 1×10¹¹M⁻¹ or greater.

Another aspect of the invention pertains to the B cells from such micewhich can be used to generate hybridomas expressing human monoclonalantibodies which bind with high affinity (e.g., greater than 2×10⁹ M⁻¹)to EGFR. Thus, in another embodiment of the invention, these hybridomasare used to generate a composition comprising an immunoglobulin havingan affinity constant (K_(A)) of at least 2×10⁹ M⁻¹ for binding EGFR,wherein said immunoglobulin comprises:

a human sequence light chain composed of (1) a light chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(L) gene segment and a humanJ_(L) segment, and (2) a light chain constant region having apolypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(L) gene segment; and

a human sequence heavy chain composed of a (1) a heavy chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(H) gene segment, optionallya D region, and a human J_(H) segment, and (2) a constant region havinga polypeptide sequence which is substantially identical to a polypeptidesequence encoded by a human C_(H) gene segment.

The development of high affinity human monoclonal antibodies againstEGFR is facilitated by a method for expanding the repertoire of humanvariable region gene segments in a transgenic mouse having a genomecomprising an integrated human immunoglobulin transgene, said methodcomprising introducing into the genome a V gene transgene comprising Vregion gene segments which are not present in said integrated humanimmunoglobulin transgene. Often, the V region transgene is a yeastartificial chromosome comprising a portion of a human V_(H) or V_(L)(V_(K)) gene segment array, as may naturally occur in a human genome oras may be spliced together separately by recombinant methods, which mayinclude out-of-order or omitted V gene segments. Often at least five ormore functional V gene segments are contained on the YAC. In thisvariation, it is possible to make a transgenic mouse produced by the Vrepertoire expansion method, wherein the mouse expresses animmunoglobulin chain comprising a variable region sequence encoded by aV region gene segment present on the V region transgene and a C regionencoded on the human Ig transgene. By means of the V repertoireexpansion method, transgenic mice having at least 5 distinct V genes canbe generated; as can mice containing at least about 24 V genes or more.Some V gene segments may be non-functional (e.g., pseudogenes and thelike); these segments may be retained or may be selectively deleted byrecombinant methods available to the skilled artisan, if desired.

Once the mouse germline has been engineered to contain a functional YAChaving an expanded V segment repertoire, substantially not present inthe human Ig transgene containing the J and C gene segments, the traitcan be propagated and bred into other genetic backgrounds, includingbackgrounds where the functional YAC having an expanded V segmentrepertoire is bred into a mouse germline having a different human Igtransgene. Multiple functional YACs having an expanded V segmentrepertoire may be bred into a germline to work with a human Ig transgene(or multiple human Ig transgenes). Although referred to herein as YACtransgenes, such transgenes when integrated into the genome maysubstantially lack yeast sequences, such as sequences required forautonomous replication in yeast; such sequences may optionally beremoved by genetic engineering (e.g., restriction digestion andpulsed-field gel electrophoresis or other suitable method) afterreplication in yeast in no longer necessary (i.e., prior to introductioninto a mouse ES cell or mouse prozygote). Methods of propagating thetrait of human sequence immunoglobulin expression, include breeding atransgenic mouse having the human Ig transgene(s), and optionally alsohaving a functional YAC having an expanded V segment repertoire. BothV_(H) and V_(L) gene segments may be present on the YAC. The transgenicmouse may be bred into any background desired by the practitioner,including backgrounds harboring other human transgenes, including humanIg transgenes and/or transgenes encoding other human lymphocyteproteins. The invention also provides a high affinity human sequenceimmunoglobulin produced by a transgenic mouse having an expanded Vregion repertoire YAC transgene. Although the foregoing describes apreferred embodiment of the transgenic animal of the invention, otherembodiments are contemplated which have been classified in fourcategories:

I. Transgenic animals containing an unrearranged heavy and rearrangedlight immunoglobulin transgene;

II. Transgenic animals containing an unrearranged heavy and unrearrangedlight immunoglobulin transgene;

III. Transgenic animal containing rearranged heavy and an unrearrangedlight immunoglobulin transgene; and IV. Transgenic animals containingrearranged heavy and rearranged light immunoglobulin transgenes.

Of these categories of transgenic animal, the preferred order ofpreference is as follows II>I>III>IV where the endogenous light chaingenes (or at least the K gene) have been knocked out by homologousrecombination (or other method) and I>II>III>IV where the endogenouslight chain genes have not been knocked out and must be dominated byallelic exclusion.

III. Bispecific/Multispecific Molecules which Bind to EGFR

In yet another embodiment of the invention, human monoclonal antibodiesto EGFR, or antigen-binding portions thereof, can be derivatized orlinked to another functional molecule, e.g., another peptide or protein(e.g., an Fab′ fragment) to generate a bispecific or multispecificmolecule which binds to multiple binding sites or target epitopes. Forexample, an antibody or antigen-binding portion of the invention can befunctionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic.

Accordingly, the present invention includes bispecific and multispecificmolecules comprising at least one first binding specificity for EGFR anda second binding specificity for a second target epitope. In aparticular embodiment of the invention, the second target epitope is anFc receptor, e.g., human FcγRI (CD64) or a human Fcα receptor (CD89).Therefore, the invention includes bispecific and multispecific moleculescapable of binding both to FcγR, FcαR or FcεR expressing effector cells(e.g., monocytes, macrophages or polymorphonuclear cells (PMNs)), and totarget cells expressing EGFR. These bispecific and multispecificmolecules target EGFR expressing cells to effector cell and, like thehuman monoclonal antibodies of the invention, trigger Fcreceptor-mediated effector cell activities, such as phagocytosis of aEGFR expressing cells, antibody dependent cell-mediated cytotoxicity(ADCC), cytokine release, or generation of superoxide anion.

Bispecific and multispecific molecules of the invention can furtherinclude a third binding specificity, in addition to an anti-Fc bindingspecificity and an anti-EGFR binding specificity. In one embodiment, thethird binding specificity is an anti-enhancement factor (EF) portion,e.g., a molecule which binds to a surface protein involved in cytotoxicactivity and thereby increases the immune response against the targetcell. The “anti-enhancement factor portion” can be an antibody,functional antibody fragment or a ligand that binds to a given molecule,e.g., an antigen or a receptor, and thereby results in an enhancement ofthe effect of the binding determinants for the F_(c) receptor or targetcell antigen. The “anti-enhancement factor portion” can bind an F_(c)receptor or a target cell antigen. Alternatively, the anti-enhancementfactor portion can bind to an entity that is different from the entityto which the first and second binding specificities bind. For example,the anti-enhancement factor portion can bind a cytotoxic T-cell (e.g.,via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1 or other immune cell thatresults in an increased immune response against the target cell).

In one embodiment, the bispecific and multispecific molecules of theinvention comprise as a binding specificity at least one antibody, or anantibody fragment thereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv,or a single chain Fv. The antibody may also be a light chain or heavychain dimer, or any minimal fragment thereof such as a Fv or a singlechain construct as described in Ladner et al. U.S. Pat. No. 4,946,778,issued Aug. 7, 1990, the contents of which is expressly incorporated byreference.

In one embodiment bispecific and multispecific molecules of theinvention comprise a binding specificity for an FcαR or an FcγR presenton the surface of an effector cell, and a second binding specificity fora target cell antigen, e.g., EGFR.

In one embodiment, the binding specificity for an Fc receptor isprovided by a human monoclonal antibody, the binding of which is notblocked by human immunoglobulin G (IgG). As used herein, the term “IgGreceptor” refers to any of the eight γ-chain genes located onchromosome 1. These genes encode a total of twelve transmembrane orsoluble receptor isoforms which are grouped into three Fcγ receptorclasses: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In onepreferred embodiment, the Fcγ receptor a human high affinity FcγRI. Thehuman FcγRI is a 72 kDa molecule, which shows high affinity formonomeric IgG (10⁸-10⁹ MA).

The production and characterization of these preferred monoclonalantibodies are described by Fanger et al. in PCT application WO 88/00052and in U.S. Pat. No. 4,954,617, the teachings of which are fullyincorporated by reference herein. These antibodies bind to an epitope ofFcγRI, FcγRII or FcγRIII at a site which is distinct from the Fcγbinding site of the receptor and, thus, their binding is not blockedsubstantially by physiological levels of IgG. Specific anti-FcγRIantibodies useful in this invention are MAb 22, MAb 32, MAb 44, MAb 62and MAb 197. The hybridoma producing MAb 32 is available from theAmerican Type Culture Collection, ATCC Accession No. HB9469. Anti-FcγRIMAb 22, F(ab′)₂ fragments of MAb 22, and can be obtained from Medarex,Inc. (Annandale, N.J.). In other embodiments, the anti-Fcγ receptorantibody is a humanized form of monoclonal antibody 22 (H22). Theproduction and characterization of the H22 antibody is described inGraziano, R. F. et al. (1995) J. Immunol. 155 (10): 4996-5002 andPCT/US93/10384. The H22 antibody producing cell line was deposited atthe American Type Culture Collection on Nov. 4, 1992 under thedesignation HA022CL1 and has the accession no. CRL 11177.

In still other preferred embodiments, the binding specificity for an Fcreceptor is provided by an antibody that binds to a human IgA receptor,e.g., an Fc-alpha receptor (FcαRI (CD89)), the binding of which ispreferably not blocked by human immunoglobulin A (IgA). The term “IgAreceptor” is intended to include the gene product of one α-gene (FcαRI)located on chromosome 19. This gene is known to encode severalalternatively spliced transmembrane isoforms of 55 to 110 kDa. FcαRI(CD89) is constitutively expressed on monocytes/macrophages,eosinophilic and neutrophilic granulocytes, but not on non-effector cellpopulations. FcαRI has medium affinity (≈5×10⁷ M⁻¹) for both IgA1 andIgA2, which is increased upon exposure to cytokines such as G-CSF orGM-CSF (Morton, H. C. et al. (1996) Critical Reviews in Immunology16:423-440). Four FcαRI-specific monoclonal antibodies, identified asA3, A59, A62 and A77, which bind FcαRI outside the IgA ligand bindingdomain, have been described (Monteiro, R. C. et al., 1992, J. Immunol.148:1764).

FcαRI and FcγRI are preferred trigger receptors for use in the inventionbecause they are (1) expressed primarily on immune effector cells, e.g.,monocytes, PMNs, macrophages and dendritic cells; (2) expressed at highlevels (e.g., 5,000-100,000 per cell); (3) mediators of cytotoxicactivities (e.g., ADCC, phagocytosis); (4) mediate enhanced antigenpresentation of antigens, including self-antigens, targeted to them.

In other embodiments, bispecific and multispecific molecules of theinvention further comprise a binding specificity which recognizes, e.g.,binds to, a target cell antigen, e.g., EGFR. In a preferred embodiment,the binding specificity is provided by a human monoclonal antibody ofthe present invention.

An “effector cell specific antibody” as used herein refers to anantibody or functional antibody fragment that binds the Fc receptor ofeffector cells. Preferred antibodies for use in the subject inventionbind the Fc receptor of effector cells at a site which is not bound byendogenous immunoglobulin.

As used herein, the term “effector cell” refers to an immune cell whichis involved in the effector phase of an immune response, as opposed tothe cognitive and activation phases of an immune response. Exemplaryimmune cells include a cell of a myeloid or lymphoid origin, e.g.,lymphocytes (e.g., B cells and T cells including cytolytic T cells(CTLs)), killer cells, natural killer cells, macrophages, monocytes,eosinophils, neutrophils, polymorphonuclear cells, granulocytes, mastcells, and basophils. Some effector cells express specific Fc receptorsand carry out specific immune functions. In preferred embodiments, aneffector cell is capable of inducing antibody-dependent cell-mediatedcytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC. Forexample, monocytes, macrophages, which express FcR are involved inspecific killing of target cells and presenting antigens to othercomponents of the immune system, or binding to cells that presentantigens. In other embodiments, an effector cell can phagocytose atarget antigen, target cell, or microorganism. The expression of aparticular FcR on an effector cell can be regulated by humoral factorssuch as cytokines. For example, expression of FcγRI has been found to beup-regulated by interferon gamma (IFN-γ). This enhanced expressionincreases the cytotoxic activity of FcγRI-bearing cells against targets.An effector cell can phagocytose or lyse a target antigen or a targetcell.

“Target cell” shall mean any undesirable cell in a subject (e.g., ahuman or animal) that can be targeted by a composition (e.g., a humanmonoclonal antibody, a bispecific or a multispecific molecule) of theinvention. In preferred embodiments, the target cell is a cellexpressing or overexpressing EGFR. Cells expressing EGFR typicallyinclude tumor cells, such as bladder, breast, colon, kidney, ovarian,prostate, renal cell, squamous cell, lung (non-small cell), and head andneck tumor cells. Other EGFR-expressing cells include synovialfibroblast cells and keratinocytes which can be used as targets in thetreatment of arthritis and psoriasis, respectively.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific or multispecific molecules of theinvention are murine, chimeric and humanized monoclonal antibodies.

Chimeric mouse-human monoclonal antibodies (i.e., chimeric antibodies)can be produced by recombinant DNA techniques known in the art. Forexample, a gene encoding the Fc constant region of a murine (or otherspecies) monoclonal antibody molecule is digested with restrictionenzymes to remove the region encoding the murine Fc, and the equivalentportion of a gene encoding a human Fc constant region is substituted.(see Robinson et al., International Patent Publication PCT/US86/02269;Akira, et al., European Patent Application 184,187; Taniguchi, M.,European Patent Application 171,496; Morrison et al., European PatentApplication 173,494; Neuberger et al., International Application WO86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988 Science240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987,J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimuraet al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

The chimeric antibody can be further humanized by replacing sequences ofthe Fv variable region which are not directly involved in antigenbinding with equivalent sequences from human Fv variable regions.General reviews of humanized chimeric antibodies are provided byMorrison, S. L., 1985, Science 229:1202-1207 and by Oi et al., 1986,BioTechniques 4:214. Those methods include isolating, manipulating, andexpressing the nucleic acid sequences that encode all or part ofimmunoglobulin Fv variable regions from at least one of a heavy or lightchain. Sources of such nucleic acid are well known to those skilled inthe art and, for example, may be obtained from 7E3, ananti-GPII_(b)III_(a) antibody producing hybridoma. The recombinant DNAencoding the chimeric antibody, or fragment thereof, can then be clonedinto an appropriate expression vector. Suitable humanized antibodies canalternatively be produced by CDR substitution U.S. Pat. No. 5,225,539;Jones et al. 1986 Nature 321:552-525; Verhoeyan et al. 1988 Science239:1534; and Beidler et al. 1988 J. Immunol. 141:4053-4060.

All of the CDRs of a particular human antibody may be replaced with atleast a portion of a non-human CDR or only some of the CDRs may bereplaced with non-human CDRs. It is only necessary to replace the numberof CDRs required for binding of the humanized antibody to the Fcreceptor.

An antibody can be humanized by any method, which is capable ofreplacing at least a portion of a CDR of a human antibody with a CDRderived from a non-human antibody. Winter describes a method which maybe used to prepare the humanized antibodies of the present invention (UKPatent Application GB 2188638A, filed on Mar. 26, 1987), the contents ofwhich is expressly incorporated by reference. The human CDRs may bereplaced with non-human CDRs using oligonucleotide site-directedmutagenesis as described in International Application WO 94/10332entitled,

Humanized Antibodies to Fc Receptors for Immunoglobulin G on HumanMononuclear Phagocytes.

Also within the scope of the invention are chimeric and humanizedantibodies in which specific amino acids have been substituted, deletedor added. In particular, preferred humanized antibodies have amino acidsubstitutions in the framework region, such as to improve binding to theantigen. For example, in a humanized antibody having mouse CDRs, aminoacids located in the human framework region can be replaced with theamino acids located at the corresponding positions in the mouseantibody. Such substitutions are known to improve binding of humanizedantibodies to the antigen in some instances. Antibodies in which aminoacids have been added, deleted, or substituted are referred to herein asmodified antibodies or altered antibodies.

The term modified antibody is also intended to include antibodies, suchas monoclonal antibodies, chimeric antibodies, and humanized antibodieswhich have been modified by, e.g., deleting, adding, or substitutingportions of the antibody. For example, an antibody can be modified bydeleting the constant region and replacing it with a constant regionmeant to increase half-life, e.g., serum half-life, stability oraffinity of the antibody. Any modification is within the scope of theinvention so long as the bispecific and multispecific molecule has atleast one antigen binding region specific for an FcγR and triggers atleast one effector function.

Bispecific and multispecific molecules of the present invention can bemade using chemical techniques (see e.g., D. M. Kranz et al. (1981)Proc. Natl. Acad. Sci. USA 78:5807), “polydoma” techniques (See U.S.Pat. No. 4,474,893, to Reading), or recombinant DNA techniques.

In particular, bispecific and multispecific molecules of the presentinvention can be prepared by conjugating the constituent bindingspecificities, e.g., the anti-FcR and anti-EGFR binding specificities,using methods known in the art and described in the examples providedherein. For example, each binding specificity of the bispecific andmultispecific molecule can be generated separately and then conjugatedto one another. When the binding specificities are proteins or peptides,a variety of coupling or cross-linking agents can be used for covalentconjugation. Examples of cross-linking agents include protein A,carbodiimide, N-succinimidyl-5-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686;Liu, M A et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Othermethods include those described by Paulus (Behring Ins. Mitt. (1985) No.78, 118-132); Brennan et al. (Science (1985) 229:81-83), and Glennie etal. (J. Immunol. (1987) 139: 2367-2375). Preferred conjugating agentsare SATA and sulfo-SMCC, both available from Pierce Chemical Co.(Rockford, Ill.).

When the binding specificities are antibodies (e.g., two humanizedantibodies), they can be conjugated via sulfhydryl bonding of theC-terminus hinge regions of the two heavy chains. In a particularlypreferred embodiment, the hinge region is modified to contain an oddnumber of sulfhydryl residues, preferably one, prior to conjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific and multispecific molecule is aMAb×MAb, MAb×Fab, Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecificand multispecific molecule of the invention, e.g., a bispecific moleculecan be a single chain molecule, such as a single chain bispecificantibody, a single chain bispecific molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific andmultispecific molecules can also be single chain molecules or maycomprise at least two single chain molecules. Methods for preparing bi-and multispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific and multispecific molecules to their specifictargets can be confirmed by enzyme-linked immunosorbent assay (ELISA), aradioimmunoassay (RIA), FACS analysis, a bioassay (e.g., growthinhibition), or a Western Blot Assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986,which is incorporated by reference herein). The radioactive isotope canbe detected by such means as the use of a γ counter or a scintillationcounter or by autoradiography.

IV. Antibody Conjugates/Immunotoxins

In another aspect, the present invention features a human anti-EGFRmonoclonal antibody, or a fragment thereof, conjugated to a therapeuticmoiety, such as a cytotoxin, a drug or a radioisotope. When conjugatedto a cytotoxin, these antibody conjugates are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine). Other examples of therapeuticcytotoxins that can be conjugated to an antibody of the inventioninclude calicheamicin and duocarmycin. An antibody of the presentinvention can be conjugated to a radioisotope, e.g., radioactive iodine,to generate cytotoxic radiopharmaceuticals for treating a EGFR-relateddisorder, such as a cancer

The antibody conjugates of the invention can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Amon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982).

V. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or a combination of humanmonoclonal antibodies, or antigen-binding portion(s) thereof, of thepresent invention, formulated together with a pharmaceuticallyacceptable carrier. In a preferred embodiment, the compositions includea combination of multiple (e.g., two or more) isolated human antibodiesor antigen-binding portions thereof of the invention. Preferably, eachof the antibodies or antigen-binding portions thereof of the compositionbinds to a distinct, pre-selected epitope of EGFR.

In one embodiment, human anti-EGFR monoclonal antibodies havingcomplementary activities are used in combination, e.g., as apharmaceutical composition, comprising two or more human anti-EGFRmonoclonal antibodies. For example, a human monoclonal antibody thatmediates highly effective killing of target cells in the presence ofeffector cells can be combined with another human monoclonal antibodythat inhibits the growth of cells expressing EGFR.

In another embodiment, the composition comprises one or a combination ofbispecific or multispecific molecules of the invention (e.g., whichcontains at least one binding specificity for an Fc receptor and atleast one binding specificity for EGFR).

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one anti-tumor agent or other conventional therapy.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,bispecific and multispecific molecule, may be coated in a material toprotect the compound from the action of acids and other naturalconditions that may inactivate the compound.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous andthe like, as well as from nontoxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of one hundredpercent, this amount will range from about 0.01 percent to aboutninety-nine percent of active ingredient, preferably from about 0.1percent to about 70 percent, most preferably from about 1 percent toabout 30 percent.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.01 to 99.5% (morepreferably, 0.1 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a compositions of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic compositions may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Thesepatents are incorporated herein by reference. Many other such implants,delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the inventioncross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhance targeted drug delivery (see, e.g., V. V. Ranade(1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties includefolate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et al.);mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety. Ina most preferred embodiment, the therapeutic compounds in the liposomesare delivered by bolus injection to a site proximal to the tumor orinfection. The composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms such as bacteria and fungi.

A “therapeutically effective dosage” preferably inhibits tumor growth byat least about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit cancer can be evaluated in an animal model system predictive ofefficacy in human tumors. Alternatively, this property of a compositioncan be evaluated by examining the ability of the compound to inhibit,such inhibition in vitro by assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound can decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe. In addition to water, the carriercan be an isotonic buffered saline solution, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyetheylene glycol,and the like), and suitable mixtures thereof. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

When the active compound is suitably protected, as described above, thecompound may be orally administered, for example, with an inert diluentor an assimilable edible carrier.

VI. Uses and Methods of the Invention

The compositions (e.g., human monoclonal antibodies to EGFR andderivatives/conjugates thereof) of the present invention have in vitroand in vivo diagnostic and therapeutic utilities. For example, thesemolecules can be administered to cells in culture, e.g., in vitro or exvivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose avariety of disorders. As used herein, the term “subject” is intended toinclude human and non-human animals. Preferred human animals include ahuman patient having disorder characterized by expression, typicallyaberrant expression (e.g., overexpression) of EGFR. For example, themethods and compositions of the present invention can be used to treat asubject with a tumorigenic disorder, e.g., a disorder characterized bythe presence of tumor cells expressing EGFR including, for example,bladder, breast, colon, kidney, ovarian, prostate, renal cell, squamouscell, lung (non-small cell), and head and neck tumor cells. The methodsand compositions of the present invention can be also be used to treatother disorders, e.g., autoimmune diseases, cancer, psoriasis, orinflammatory arthritis, e.g., rheumatoid arthritis, systemic lupuserythematosus-associated arthritis, or psoriatic arthritis. The term“non-human animals” of the invention includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dog, cow,chickens, amphibians, reptiles, etc.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can be initially tested for binding activityassociated with therapeutic or diagnostic use in vitro. For example,compositions of the invention can be tested using the ELISA and flowcytometric assays described in the Examples below. Moreover, theactivity of these molecules in triggering at least one effector-mediatedeffector cell activity, including cytolysis of cells expressing EGFR canbe assayed. Protocols for assaying for effector cell-mediatedphagocytosis are described in the Examples below.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention have additional utility in therapy anddiagnosis of EGFR-related diseases. For example, the human monoclonalantibodies, the multispecific or bispecific molecules can be used, forexample, to elicit in vivo or in vitro one or more of the followingbiological activities: to opsonize a cell expressing EGFR; to mediatephagocytosis or cytolysis of a cell expressing EGFR in the presence ofhuman effector cells; to inhibit EGF or TGF-α inducedautophosphorylation in a cell expressing EGFR; to inhibit autocrine EGFor TGF-α-induced activation of a cell expressing EGFR; or to inhibit thegrowth of a cell expressing EGFR, e.g., at low dosages.

In another embodiment, the human monoclonal antibodies of the presentinvention are unable to induce complement-mediated lysis of cells and,therefore, has fewer side effects in triggering complement-activatedafflictions, e.g., acne. The primary cause of acne is an alteration inthe pattern of keratinization within the follicle that produce sebum.Since keratinocytes express EGFR, interference with EGFR signalingprocesses in the skin can alter the growth and differentiation of thekeratinocytes in the follicles which results in the formation of acne.Direct immunofluorescent studies have shown that in early non-inflamedand inflamed acne lesions there is activation of the classical andalternative complement pathways.

In a particular embodiment, the human antibodies and derivatives thereofare used in vivo to treat, prevent or diagnose a variety of EGFR-relateddiseases. Examples of EGFR-related diseases include a variety ofcancers, such as bladder, breast, uterine/cervical, colon, pancreatic,colorectal, kidney, stomach, ovarian, prostate, renal cell, squamouscell, lung (non-small cell), esophageal, and head and neck cancer.

In another aspect the invention relates to a method of treating orpreventing psoriasis, rheumatoid arthritis, systemic lupuserythematosus, psoriatic arthritis, Menetrier's disease, systemicsclerosis, Sjögren's syndrome, pulmonary fibrosis, bronchial asthma,myelofibrosis, diabetic nephropathy, chronic allograft rejection,chronic glomerulonephritis, Crohn's disease, ulcerative colitis, hepaticcirrhosis, sclerosing cholangitis, chronic uveitis, or cicatricialpemphigoid, as well as methods of treating or preventing Alzheimer'sdisease or other forms of dementia.

Methods of administering the compositions (e.g., human antibodies,multispecific and bispecific molecules) of the invention are known inthe art. Suitable dosages of the molecules used will depend on the ageand weight of the subject and the particular drug used. The moleculescan be coupled to radionuclides, such as 131I, 90Y, 105Rh, indium-111,etc., as described in Goldenberg, D. M. et al. (1981) Cancer Res. 41:4354-4360, and in EP 0365 997. In another aspect the invention relatesto an immunoconjugate comprising an antibody according to the inventionlinked to a radioisotope, cytotoxic agent (e.g., calicheamicin andduocarmycin), a cytostatic agent, or a chemotherapeutic drug. Thecompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be coupled to anti-infectiousagents.

In another embodiment, the human anti-EGFR antibodies, or antigenbinding fragments thereof, can be co-administered with a therapeuticagent, e.g., a chemotherapeutic agent, an immunosuppressive agent, anant-inflammatory agent, or an ant-psoriasis agent, or can beco-administered with other known therapies, such as physical therapies,e.g., radiation therapy, hyperthermia, transplantation (e.g., bonemarrow transplantation), surgery, sunlight, or phototherapy. Suchtherapeutic agents include, among others, anti-neoplastic agents such asdoxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine,chlorambucil, and cyclophosphamide hydroxyurea which, by themselves, areonly effective at levels which are toxic or subtoxic to a patient.Cisplatin is intravenously administered as a 100 mg/m² dose once everyfour weeks and adriamycin is intravenously administered as a 60-75 mg/m²dose once every 21 days.

Pharmaceutical compositions of the present invention can include one ormore further chemotherapeutic agents selected from the group consistingof nitrogen mustards (e.g., cyclophosphamide and ifosfamide), aziridines(e.g., thiotepa), alkyl sulfonates (e.g., busulfan), nitrosoureas (e.g.,carmustine and streptozocin), platinum complexes (e.g., carboplatin andcisplatin), non-classical alkylating agents (e.g., dacarbazine andtemozolamide), folate analogs (e.g., methotrexate), purine analogs(e.g., fludarabine and mercaptopurine), adenosine analogs (e.g.,cladribine and pentostatin), pyrimidine analogs (e.g., fluorouracil(alone or in combination with leucovorin) and gemcitabine), substitutedureas (e.g., hydroxyurea), antitumor antibiotics (e.g., bleomycin anddoxorubicin), epipodophyllotoxins (e.g., etoposide and teniposide),microtubule agents (e.g., docetaxel and paclitaxel), camptothecinanalogs (e.g., irinotecan and topotecan), enzymes (e.g., asparaginase),cytokines (e.g., interleukin-2 and interferon-α), monoclonal antibodies(e.g., trastuzumab and bevacizumab), recombinant toxins and immunotoxins(e.g., recombinant cholera toxin-B and TP-38), cancer gene therapies,physical therapies (e.g., hyperthermia, radiation therapy, and surgery)and cancer vaccines (e.g., vaccine against telomerase).

In another aspect the pharmaceutical composition comprises one or morefurther therapeutic agents selected from the group consisting ofimmunosuppressive antibodies (e.g., antibodies against MHC, CD2, CD3,CD4, CD7, CD28, B7, CD40, CD45, IFN-γ TNF-α, IL-4, IL-5, IL-6R, IL-7,IL-8, IL-10, CD11a, CD20, or CD58, or antibodies against their ligands)and other immunomodulatory compounds (e.g., soluble IL-15R or IL-10).

In another aspect the pharmaceutical composition comprises one or morefurther immunosuppressive agents selected from the group consisting ofcyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil,corticosteroids (e.g., prednisone), methotrexate, gold salts,sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine,15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin,tacrolimus (FK-506), OKT3, anti-thymocyte globulin.

In another aspect the pharmaceutical composition comprises one or morefurther anti-inflammatory agents selected from the group consisting ofaspirin and other salicylates, steroidal drugs, NSAIDs (nonsteroidalanti-inflammatory drugs) (e.g., ibuprofen, fenoprofen, naproxen,sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone,etodolac, oxaprozin, and indomethacin), Cox-2 inhibitors (e.g.,rofecoxib and celecoxib), and DMARDs (disease modifying antirheumaticdrugs) (e.g., methotrexate, hydroxychloroquine, sulfasalazine,azathioprine, pyrimidine synthesis inhibitors (e.g., leflunomide), IL-1receptor blocking agents (e.g., anakinra), TNF-α, blocking agents (e.g.,etanercept, infliximab and adalimumab), anti-IL-6R antibodies, CTLA4Ig,and anti-IL-15 antibodies).

In another aspect the pharmaceutical composition comprises one or morefurther anti-psoriasis agents selected from the group consisting of coaltar, A vitamin, anthralin, calcipotrien, tarazotene, corticosteroids,methotrexate, retinoids (e.g., acitretin), cyclosporine, etanercept,alefacept, efaluzimab, 6-thioguanine, mycophenolate mofetil, tacrolimus(FK-506), and hydroxyurea.

Co-administration of the human anti-EGFR antibodies, or antigen bindingfragments thereof, of the present invention with chemotherapeutic agentsprovides two anti-cancer agents which operate via different mechanismswhich yield a cytotoxic effect to human tumor cells. Suchco-administration can solve problems due to development of resistance todrugs or a change in the antigenicity of the tumor cells which wouldrender them unreactive with the antibody.

Target-specific effector cells, e.g., effector cells linked tocompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be used as therapeutic agents.Effector cells for targeting can be human leukocytes such asmacrophages, neutrophils or monocytes. Other cells include eosinophils,natural killer cells and other IgG- or IgA-receptor bearing cells. Ifdesired, effector cells can be obtained from the subject to be treated.The target-specific effector cells, can be administered as a suspensionof cells in a physiologically acceptable solution. The number of cellsadministered can be in the order of 10⁸-10⁹ but will vary depending onthe therapeutic purpose. In general, the amount will be sufficient toobtain localization at the target cell, e.g., a tumor cell expressingEGFR, and to effect cell killing by, e.g., phagocytosis. Routes ofadministration can also vary.

Therapy with target-specific effector cells can be performed inconjunction with other techniques for removal of targeted cells. Forexample, anti-tumor therapy using the compositions (e.g., humanantibodies, multispecific and bispecific molecules) of the inventionand/or effector cells armed with these compositions can be used inconjunction with chemotherapy. Additionally, combination immunotherapymay be used to direct two distinct cytotoxic effector populations towardtumor cell rejection. For example, anti-EGFR antibodies linked toanti-Fc-gammaRI or anti-CD3 may be used in conjunction with IgG- orIgA-receptor specific binding agents.

Bispecific and multispecific molecules of the invention can also be usedto modulate FcαR or FcγR levels on effector cells, such as by cappingand elimination of receptors on the cell surface. Mixtures of anti-Fcreceptors can also be used for this purpose.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention which have complement binding sites, such asportions from IgG1, -2, or -3 or IgM which bind complement, can also beused in the presence of complement. In one embodiment, ex vivo treatmentof a population of cells comprising target cells with a binding agent ofthe invention and appropriate effector cells can be supplemented by theaddition of complement or serum containing complement. Phagocytosis oftarget cells coated with a binding agent of the invention can beimproved by binding of complement proteins. In another embodiment targetcells coated with the compositions (e.g., human antibodies,multispecific and bispecific molecules) of the invention can also belysed by complement. In yet another embodiment, the compositions of theinvention do not activate complement.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be administered together withcomplement. Accordingly, within the scope of the invention arecompositions comprising human antibodies, multispecific or bispecificmolecules and serum or complement. These compositions are advantageousin that the complement is located in close proximity to the humanantibodies, multispecific or bispecific molecules. Alternatively, thehuman antibodies, multispecific or bispecific molecules of the inventionand the complement or serum can be administered separately.

Compositions of the present invention can also include an expressionvector comprising a nucleotide sequence encoding the variable region ofa light chain, heavy chain or both light and heavy chains of a humanantibody which binds EGFR, and further comprising a nucleotide sequenceencoding the constant region of a light chain, heavy chain or both lightand heavy chains of a human antibody which binds EGFR. In a particularembodiment, the invention relates to a pharmaceutical compositioncomprising an expression vector comprising a nucleotide sequenceencoding heavy chain and light chain variable regions which comprise theamino acid sequences shown in SEQ ID NO:2 and SEQ ID NO:4, respectively,and conservative sequence modifications thereof.

Also within the scope of the invention are kits comprising thecompositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention and instructions for use. The kit canfurther contain a least one additional reagent, such as complement, orone or more additional human antibodies of the invention (e.g., a humanantibody having a complementary activity which binds to an epitope inEGFR antigen distinct from the first human antibody).

In other embodiments, the subject can be additionally treated with anagent that modulates, e.g., enhances or inhibits, the expression oractivity of Fcα or Fcγ receptors by, for example, treating the subjectwith a cytokine. Preferred cytokines for administration during treatmentwith the multispecific molecule include of granulocytecolony-stimulating factor (G-CSF), granulocyte-macrophagecolony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), and tumornecrosis factor (TNF).

In another embodiment, the subject can be additionally treated with alymphokine preparation. Cancer cells which do not highly express EGFRcan be induced to do so using lymphokine preparations. Lymphokinepreparations can cause a more homogeneous expression of EGFRs amongcells of a tumor which can lead to a more effective therapy. Lymphokinepreparations suitable for administration include interferon-gamma, tumornecrosis factor, and combinations thereof. These can be administeredintravenously. Suitable dosages of lymphokine are 10,000 to 1,000,000units/patient.

The compositions (e.g., human antibodies, multispecific and bispecificmolecules) of the invention can also be used to target cells expressingFcγR or EGFR, for example for labeling such cells. For such use, thebinding agent can be linked to a molecule that can be detected. Thus,the invention provides methods for localizing ex vivo or in vitro cellsexpressing Fc receptors, such as FcγR, or EGFR. The detectable label canbe, e.g., a radioisotope, a fluorescent compound, an enzyme, or anenzyme co-factor.

In one embodiment, the invention provides methods for detecting thepresence of EGFR antigen in a sample, or measuring the amount of EGFRantigen, comprising contacting the sample, and a control sample, with ahuman monoclonal antibody, or an antigen binding portion thereof, whichspecifically binds to EGFR, under conditions that allow for formation ofa complex between the antibody or portion thereof and EGFR. Theformation of a complex is then detected, wherein a difference complexformation between the sample compared to the control sample isindicative the presence of EGFR antigen in the sample.

In still another embodiment, the invention provides a method fordetecting the presence or quantifying the amount of Fc-expressing cellsin vivo or in vitro. The method comprises (i) administering to a subjecta composition (e.g., a multi- or bispecific molecule) of the inventionor a fragment thereof, conjugated to a detectable marker; (ii) exposingthe subject to a means for detecting said detectable marker to identifyareas containing Fc-expressing cells.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

EXAMPLES Materials and Methods Antigen:

Transgenic mice were immunized with the A431 Human epidermoid carcinomacell line (CRL-1555, Lot 203945, ATCC Manassas, Va.) and with solubleepidermal growth factor receptor (EGFR) obtained from Sigma Chemical Co(product E 3641 lot 109H4108 and 20K4079). Soluble EGFR was stored at−20° to −80° C. until use.

Media Formulations:

(A) High Glucose DMEM (Mediatech Cellgro #10013) containing 10% FBS,Pennicillin-Streptomycin (Sigma P-7539), and 2-mercaptoethanol (GibcoBRL21985-023) was used to culture A431 cells and myeloma cells. Additionalmedia supplements were added to the Hybridoma growth media, whichincluded: Origin-Hybridoma Cloning Factor (Igen 21001), OPI supplement(Sigma O-5003), HAT or HT (Sigma H 0262, H 0137). (B) Serum Free Mediumcontains DMEM, antibiotics and 2-mercaptoethanol only.

Cells for Immunization:

Cells for immunization were grown in DMEM (see above) to confluence onT-75 cell culture flasks, and were harvested with Trypsin EDTA(Cellgrow, Cat #25-053-Cl) solution 5-10 ml per flask. Cells recoveredfrom flasks were resuspended in 50 ml of complete medium and then washedby three cycles of centrifugation (1000 G) and resuspended in 50 ml ofsterile PBS. Mice were injected with 1×10⁷ cells suspended in 0.5 ml ofsterile PBS.

EGFR:

Soluble EGFR was mixed with Ribi adjuvant (Sigma, M 6536) in sterile PBSat a concentration of 25 μg EGFR/100 μl. Final tail vein immunizationswere performed with soluble EGFR in sterile PBS.

Transgenic Mice:

Mice were housed in filter cages and were evaluated to be in goodphysical condition on the date of the fusion. Mice that produced theselected hybridomas were males 6-8 weeks old of the (CMD)++;(HCo7)11952+; (JKD)++; (KCoS)9272+genotype (see Table 1).

TABLE 1 Genotype Data* Mouse Sex Born Genotype 20241 Male Sep. 21, 1999CMD++ (HCo7) 11952 + (JKD) ++ (KCo5) 9272 20242 Male Sep. 21, 1999 CMD++(HCo7) 11952 + (JKD) ++ (KCo5) 9272 20243 Male Sep. 21, 1999 CMD++(HCo7) 11952 + (JKD) ++ (KCo5) 9272 *Individual transgene designationsare in parentheses, followed by line numbers for randomly integratedtransgenes. The symbol ++ and + indicate homozygous or hemizygous;however, because the mice are routinely screened using a PCR-based assaythat does not allow us to distinguish between heterozygosity andhomozygosity for the randomly integrated human Ig transgenes, a +designation may be given to mice that are actually homozygous for theseelements.

Antibodies:

The following anti-EGFR MAbs were used in vitro and in vivo: 2F8 (alsoreferred to as “Humax-EGFR”), a human IgG1 anti-EGFR antibody (Genmab,Utrecht, The Netherlands); the hybridoma producing m225, a mouse IgG2aanti-EGFR antibody, was obtained from American Type Culture Collection(ATCC, Rockville, Md., HB-8508); irrelevant human IgG isotype control(Genmab) which was used as an irrelevant IgG1 antibody; and fluoresceinisothiocynate (FITC)-conjugated F(ab′)₂ fragment of goat anti-mouse IgG(H+L) which was used as the secondary antibody for indirectimmunofluorescence (Protos, San Francisco, Calif.), FITC-conjugatedF(ab′)₂ rabbit-a-human IgG (DAKO, Glostrup, Denmark).

The 2F8 hybridoma was cultured in DMEM (Gibco BRL, Life Technologies,Paisley, Scotland) supplemented with 10% Fetal bovine serum (FBS)(Hyclone, Logan, Utah) and 100 U/ml penicillin and 100 U/ml streptomycin(both Gibco BRL) (pen/strep). The m225 hybridoma was cultured inRPMI1640 (Gibco BRL) supplemented with 15% FBS (Hyclone) and pen/strep(both Gibco BRL). All cell lines were kept at 37° C. in humidifiedatmosphere containing 5% carbon dioxide. Humax antibody was purifiedusing protein-A affinity chromatography followed by size exclusion on aHR200 column (Pharmacia, New Jersey). Mouse antibodies were purifiedusing protein-G chromatography followed by size exclusion on a HR200column. The purity of all antibodies was >95% as determined by dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). F(ab′) fragmentswere made via pepsin or β-mercaptoethanol treatment followed byprotein-A/G purification. Isolated F(ab′) fragments were >95% pure asdetermined by SDS-PAGE.

Cell Lines:

A431, an epidermoid carcinoma which highly over-expresses EGFR, wasobtained from the ATCC (Rockville, Md., CRL-155). The cells werecultured in RPMI 1640 medium (Gibco BRL), supplemented with 10%heat-inactivated FBS (Hyclone), 50 μg/ml streptomycin, 50 IU/mlpenicillin, and 4 mM L-glutamine (all Gibco BRL). As the cells growadherent they were detached by using trypsin-EDTA in PBS (LifeTechnologies, Paisley, Scotland). In tumor models, the cells are alwaysused in log-phase. The cells are tested for stable EGFR expression andmycoplasma contamination before each experiment.

Fusion Procedure:

Spleens were aseptically harvested from freshly euthanized mice, andplaced in 20-30 ml cold serum free media (SFM) in a petri plate.Adherent tissue was removed and spleens were rinsed twice in SFM. Spleencells were gently harvested by homogenization in a tissue grinder inSFM.

Cells were centrifuged at 1000 g for 10 minutes and the red blood cellsin the cell pellet were lysed by suspending the spleen cell pellet in 5ml of ice cold 0.17 M NH₄Cl for 2-5 minutes. The cell mix was thendiluted with 20 ml of SFM and centrifuged at 1000 g for 10 minutes.Myeloma cells were harvested into 50 ml centrifuge tubes. Spleen cellsand myeloma cells were then washed by three cycles of centrifugation at1000 g and resuspended in 30-40 ml of SFM.

After a cell count, spleen and myeloma cells were mixed at a 1:1 to 4:1ratio spleen/myeloma. The spleen cell/myeloma cell mix was pelleted bycentrifugation and the supernatant was removed by aspiration. The fusionwas done by adding 1-2 ml of PEG solution (Sigma # P-7181) drop wise tothe cell pellet over 45 seconds and gently mixing the solution for 75seconds. The PEG solution was slowly diluted by adding 2 ml SFM dropwise over a minute. This was repeated with another 2 ml of SFM and thenthe solution was allowed to stand for 1 minute.

The solution was then slowly diluted with an additional 30 ml of SFMover 90 seconds. Cells were centrifuged at 1000 g and for 10 minutes andresuspended in 30 Ml of HAT medium. The fusion mix was diluted to200-300 ml in HAT supplemented medium containing 3% Origin HybridomaCloning Factor, and dispersed into 96 well plates at >200 μl/well(˜10-15 plates/spleen). Hybridoma plates were examined after 3-7 daysfor hybridomas. Plates were fed at 7 days by replacing half the mediumin each well with fresh HT medium supplemented with 3% Origin. Plateswere fed every 3-4 days thereafter with HT Medium.

ELISA Reagents:

1. Phosphate buffered saline (PBS), D-PBS without Ca and Mg, HycloneD-PBS # SH30013.03, or Sigma P 3813.2. PBS-T (wash buffer), PBS containing 0.05% Tween 20, Sigma P-1379.3. PBS-T plus 1% BSA (Sigma A 9647). This serves as the blocking bufferand sample buffer.4. ELISA plates, Nunc Immuno-plate F96 Maxisorp 442404.5. Anti-human IgG γ-chain specific antibody, Jackson ImmunoResearch#109-006-098.6. Alkaline phosphatase labeled Goat anti-human γ-chain specific IgG,Jackson Immuno Research #109-056-098. An alternate is to use alkalinephosphatase labeled anti-human κ, Sigma A 3813.7. Alkaline phosphatase labeled anti-human IgG1, or IgG3, SouthernBiotechnology #9050-04 & 9210-04, for use in isotype specific ELISA.8. p nitrophenyl (pNPP), Sigma N2765, or Sigma Fast tablet kit N-2770.9. pNPP Substrate and buffer—Two options:A. Diethanolamine buffer: Mix 97 ml of diethanolamine, Sigma D-2286,plus 0.1 g MgCl₂6H₂O and 800 ml Di Water. Adjust the pH to 9.8 andadjust final volume to 1.0 L with Di water. Add one 20 mg tablet ofpNPP, Sigma N-2765, per 20 ml diethanolamine buffer.B. Sigma Fast pNPP Tablet Set, Sigma N-2770: Dissolve 1 buffer tabletand 1 pNPP tablet in 20 ml H₂O.10. ELISA plate reader with 405 nm filter.11. Epidermal growth factor receptor (EGFR), Sigma E 3641, Biotinlabeled EGF (EGF-B), Molecular Probes E-3477.12. Biotin labeled anti EGFR MAbs or human antibodies.13. Nonspecific human antibodies for negative controls, or purifiedhuman IgG1 κ, Sigma 1-13889.14. Automated ELISA plate washer: Titertek MAP C.

Anti Human IgG, κ ELISA:

To screen hybridoma plates for human IgG, κ producing MAbs, ELISA plateswere coated with 1 μg/ml of anti-human IgG γ-chain specific antibody,Jackson ImmunoResesarch #109-006-098, overnight or longer at 4° C.Plates were washed in a plate washer and 100 μl/well PBS-T plus 1% BSAwas added. Plates were incubated at least 15 minutes and 10-50 μl ofcell culture supernatant was added to ELISA plate wells with a few wellsin each plate included with IgG as a positive control and cell culturemedium as a negative control. Plates were incubated 1-2 hr at roomtemperature, washed, and alkaline phosphatase labeled anti-Human κantibody (Sigma A-3813) 1:5000 in PBS-T plus 1% BSA was added. Plateswere incubated for 1 hr at room temperature, washed 4 times in a platewasher, and pNPP substrate was added. Plates were incubated 10-60minutes and absorbance was read at 405 nm in an ELISA plate reader.

ELISA Procedure for Testing Specificity of Anti-EGFR HumanAntibodies—Direct Binding of Antibody to EGFR Coated ELISA Plates:

To verify that anti-EGFR antibodies specifically bind to EGFR, NuncMaxisorp plates were coated with 100 μl/well of EGFR at 0.4 μg/ml in PBSovernight at 4° C. or for 2 hr at room temperature. Plates were washedin PBS-T three times, 100 μl/well of PBS-T plus 1% BSA was added toblock nonspecific sites on the plastic surface, and incubated at least15 minutes before loading samples. Dilutions of samples to be testedwere loaded in PBS-T plus 1% BSA. Supernatants were diluted a minimum of1:3 in PBS-T 1% BSA for loading into ELISA plates. Samples and standardswere loaded at 100 μl well, incubated for 1 hr at room temperature, andplates were washed three times in PBS-T. 100 μl/well of PBS-T 1% BSAcontaining alkaline phosphatase labeled goat antihuman γ specificantibody at a 1:3000 to 1:5000 dilution was added. Alternatively,alkaline phosphatase labeled anti-human κ can be used. Plates wereincubated 1 hr at room temp, washed 4 times, and pNPP substrate wasadded. A absorbance was read at 405 nm.

ELISA Procedure for Testing Specificity of anti-EGFR HumanAntibodies—ELISA EGF/EGFR Blocking Assay:

To verify that anti-EGFR antibody binds to EGFR and additionally blocksthe binding of biotin labeled epidermal growth factor to the Epidermalgrowth factor receptor (EGFR), Nunc Maxisorp plates were coated with 100μl/well of EGFR at 0.4 μg/ml in PBS overnight at 4° C. or for 2 hr atroom temperature. Plates were washed in PBS-T three times, 100 μl/wellof PBS-T plus 1% BSA was added to block nonspecific sites on the plasticsurface, and incubated at least 15 minutes before loading samples.Dilutions of samples to be tested were loaded in PBS-T plus 1% BSA.Supernatants were diluted a minimum of 1:3 in PBS-T 1% BSA for loadinginto ELISA plates. Samples and standards were loaded at 100 μl well,incubated for 30 minutes at room temperature, and 20 μl/well of Biotinlabeled EGF at 0.5 μg/ml was added and plates were incubated for 1 hr(this is added to the sample solution already on the plates).Alternatively, samples can be incubated for 1 hr, washed, and 100μl/well EGF-biotin at 0.1 μg/ml added and incubated for 1 hr. Plateswere washed 3 times, 100 μl/well of PBS-T 1% BSA containing streptavidinalkaline phosphatase at 1:2000 dilution was added, and incubated 1 hr.Plates were washed 4 times, pNPP substrate was added, and absorbance wasread at 405 nm.

Competitive ELISA for Determining Epitope Specificity of Anti-EGFR HumanAntibodies—Competition with Commercial Murine MAbs 225, 528, AB5, and29.1:

This assay was performed to determine which MAbs are most likeantibodies 225, 528, AB5 and 29.1. MAbs 225, 528, and AB5 block EGFbinding to its receptor and inhibit in-vivo endogenous tyrosine kinaseactivity of EGFR. MAb 29.1 is a non blocking MAb that binds to acarbohydrate residue of EGFR. Plates were coated for at least 2 hr atroom temperature, or overnight at 4° C., with 0.4 μg/ml of EGFR in PBSand washed and blocked with 100 μl/well of PBS-T 1% BSA. The blockingsolution was flicked out and 100 μl/well of PBS-T-1% BSA was added tocolumns 1-6 on the left side of the plate while an unlabeled mouse MAbat 1 μg/ml (100 μl/well) was added to the right side of the plate incolumns 7-12. Plates were incubated at room temperature for 1 hour and25 μl of cell culture supernatant was added to the equivalent positionof each half of the plate so that each supernatant is loaded onto onewell with PBS-T-1% BSA and one well with mouse MAb. Plates wereincubated 1 hr, washed, and alkaline phosphatase labeled anti-Human IgGFc antibody was added. Plates were incubated 1 hr. Plates were washedand substrate was added. Absorbence was read at 405 nm. The %competition from MAb was determined by the following formula: (ODsupernatant without competition−OD supernatant with MAb competition/ODsupernatant without competition)×100.

Competitive ELISA for Determining Epitope Specificity of Anti-EGFR HumanAntibodies—Competitive ELISA with Biotin Labeled Human Antibodies:

Competitive ELISA assays were also performed to determine thespecificity of the anti-EGFR human antibodies. Plates were coated for atleast 2 hr at room temperature, or overnight at 4° C., with 0.4 μg/ml ofEGFR in PBS. Plates were washed and blocked with 100 μl/well of PBS-T 1%BSA. 50 μl of (10-30 μg/ml) of unlabeled human antibodies or mouse MAbswas added to the top well(s) of the plate column and 50 μl wassequentially transferred and mixed serially down each column to create athree fold dilution series of each antibody. 50 μl was discarded fromthe bottom well after mixing. Plates were incubated for 1 hr and 20μl/well of biotinylated anti-EGFR human antibody or unlabeled mouseanti-Human EGFR antibodies was added to the entire plate so that thefinal concentration of competing antibody was approximately 0.1-0.2μg/ml. Plates were incubated for 1 hour at room temperature, washed, and100 μl/well streptavidin alkaline phosphatase (1:2000 in PBS-T-BSA) orAlkaline phosphatase labeled goat anti-Mouse IgG was added. Plates wereincubated 1 hour, washed, and substrate was added. Absorbence was readat 405 nm.

FACS Procedure for Testing Specificity of Anti-EGFR HumanAntibodies—EGF/EGFR Blocking:

This assay was used to verify that anti-EGFR antibody binds to EGFR onthe cell surface, and by doing so, blocks the binding of biotin labeledepidermal growth factor (EGF-B) to the Epidermal growth factor receptor(EGFR). This FACS based method uses the human epidermal carcinoma cellline A431 which expresses about 10⁶ EGFR molecules/cell.

Materials for EGFR FACS Assays:

1. A431 cells (ATCC CRL 1555) confluent in one or more T-175 flasks. A431 cells are cultured in DMEM plus 10% FCS.2. Trypsin-EDTA solution, Sigma T-3924.3. Biotin labeled EGF. Prepare a stock solution of about 5 μg/ml, use 10μl/well.4. Round bottomed 96 well plates.5. PBS, sterile.6. PBS plus 1% BSA plus 0.02% sodium azide (FACS buffer).7. PE-labeled Streptavidin, Sigma S 3402. Dilute 1:20 in FACS buffer.8. PE-labeled or FITC labeled anti human IgG, FC γ specific, Pharminigen34164X, 34165X.9. Low speed centrifuge with swinging buckets and adapter for 96 wellplates (Beckman).

10. FACS

11. BD FACS tubes.Procedure: A431 cells were harvested by trypsin EDTA treatment. Mediumfrom tissue culture flask was removed and flask was rinsed briefly with10-20 ml sterile PBS or HBSS. 5-10 ml of trypsin EDTA was added andflask was returned to incubator for a few minutes. As cells began todetach from the plastic surface, a 10 ml pipette was used to gentlysyringe the cells from the plastic surface and to generate a single cellsuspension without too many cell clumps. Cells were transferred to a 50ml tube with 20-30 ml of cell culture medium (with FBS), centrifuged for10 min at 1000 g, and washed twice by centrifugation and resuspension ofcells in cold FACS buffer. Cell solution was filtered through a nylonmesh to remove cell clumps (the top of BD FACS tubes are equipped forthis). Cells were counted and the volume was adjusted so that there arebetween 1 to 5×10⁶ cells ml. Cells were dispensed into a round bottom 96well plate at about 200,000 cells/well and centrifuged for about 1 minat 1000 g and then the liquid was flicked out (cells should remain inwell bottom). Plates were kept on ice or at 4° C. In a separate 96-wellplate, antibody sample dilutions in FACS buffer was prepared bypreparing a three fold dilution series of antibody starting at 10 μg/mland decreasing to 4.5 ng/ml. 100 μl of each antibody dilution, isotypecontrols, and buffer controls was added to the round bottom plate. Theantibody samples and controls were mixed with the cells and incubatedfor 30 minutes on ice. 10 μl of biotin labeled EGF was added to theantibody cell solution and incubated an additional 30 minutes. The cellswere washed three times by centrifugation and resuspension in FACSbuffer. 50 μl well of Streptavadin PE was added, mixed, and incubatedfor 30 minutes on ice. The cells were washed three times and resuspendedin 50 μl FACS buffer. The contents of each well were transferred to atube containing 300-400 μl FACS buffer. 5000-10000 cells were analyzedin each sample by FACS in the FL-2 channel. MCF versus Antibodyconcentration was plotted.

Human or Animal Derived Materials:

A431 Human epidermoid carcinoma cell line (CRL-1555, Lot 203945, ATCCManassas, Va.). Trypsin EDTA (Cellgrow Cat #25-053-Cl). P3 X63 ag8.653myeloma cell line: ATCC CRL 1580, lot F-15183 Origin-Hybridoma CloningFactor (Igen 21001). OPI supplement (Sigma O-5003) Fetal bovine serum(SH30071 lot #s ALE10321, and AGH6843) from Hyclone, Logan, Utah. OrigenFreeze Medium (Igen, #210002)

ELISA:

For determining the binding of human antibodies to EGFR, an ELISA withEGFR (Sigma, St Louis, M) coated overnight in a concentration of 1 μg/mlin PBS on a 96-wells microtiter plate (Greiner, Frickenhausen, Germany)was used. After blocking the plate with ELISA buffer (PBS/0.05% Tween 20and 1% chicken serum (Gibco BRL)) at a concentration of 100 μl/well,monoclonal antibody diluted in ELISA buffer was added and incubated for1 hour at 37° C. The plates were subsequently washed 3 times andincubated with peroxidase labeled goat anti-human IgG Fc specific(Jackson, West Grace, P) for 1 hour at 37° C. The assay was developedwith ABTS (Roche Diagnostics, Mannheim, Germany) for 30 minutes.Absorbance was measured with a microplate reader (Biotek, Winooski,Canada) at 415 nm. With regard to blocking studies, the plates werepre-incubated for 10 minutes with 50 μl blocking agent in ELISA bufferbefore adding 50 μl fully human antibody. For determining human IgG inmouse serum ELISA plates were coated with rabbit anti-human kappa, lightchains (DAKO) overnight in PBS in a 96-wells microtiter plate (Greiner).After blocking the plate with ELISA buffer (PBS/0.05% Tween20 and 1%chicken serum) 100 μl/well, mouse serum diluted in ELISA buffer wasadded and incubated for 1 hour at 37° C. The plates were subsequentlywashed 3 times and incubated with peroxidase labeled rabbit F(ab′)₂fragments anti human IgG (DAKO) for 1 hour at 37° C. The assay wasdeveloped with ABTS (Roche) for 30 minutes. Absorbance was measured witha microplate reader (Biotek) at 415 nm.

Flow Cytometry:

EGFR over expressing tumor cells were incubated with MAb for 30 minutesat 4° C. Cells were washed three times in phosphate buffered salinesupplemented with 1% bovine serum albumin (Roche) and 0.01% azide.Counter-staining was performed with FITC-conjugated F(ab′)₂ fragments ofa goat anti-mouse antibody or with FITC-conjugated F(ab′)₂ fragments ofa rabbit anti-human IgG antibody. With regard to inhibition experiments,the cells were pre incubated with EGF or TGF-α, for 10 minutes at 4° C.All samples were analyzed on a FACScan flowcytometer (Becton-Dickinson,San Jose, Calif.).

Phosphorylation Studies:

Sub-confluent cultures of A431 cells in 24-wells plates (NUNC, Kamstrup,Denmark) were treated overnight with low serum conditions (0.5%).Different antibody dilutions were added to the wells and incubated for30 minutes at 37° C. and 5% carbon dioxide. Cells were stimulated withor without 5 ng/ml EGF (Prepotech, Rocky Hill, N.J.) for 5 min at 37° C.and 5% carbon dioxide. Cell extracts were prepared as described by Tomicet al. (Tomic et al, 1995) using 100 μl of lysis buffer per well. Fiftyμl of A431 cell extract was analyzed by sodium SDS-PAGE andimmunoblotting with anti-phospho-tyrosine antibodies (PY20, TransductionLaboratories, Kentucky), goat anti mouse IgG-HRP antibodies(Transduction Laboratories), and ECL detection. For stimulation withTGF-α (Prepotech, Rocky Hill, N.J.) sub-confluent cultures of A431 cellsin 24 well plates (Nunc) were treated overnight with low-serum medium(0.5%). Antibodies were added in a fixed dose of 10 or 0 μg/ml andincubated as described as above. The cells were stimulated with anincreasing amount TGF-α. Cells were treated as above.

In Vitro Cell Growth Inhibition:

Cell growth inhibition features of fully human antibodies were evaluatedwith a non-radioactive inhibition assay. Briefly, 100 μl of 2×10⁴/mlA431 cells was added to flat-bottomed tissue culture plates and placedin a cell culture incubator. After 2 hours 100 μl antibody dilution wasadded and placed back in the cell culture incubator. The cells wereincubated for 6-7 days, supernatants were decanted, and 100 μl 0.25%glutaraldehyde in PBS was added to each well. After incubation for 45minutes at room temperature the wells were washed two times withdemi-water. 50 μl of 1% crystal violet in demi-water was added andincubated for 15 minutes at room temperature. After washing the platetwice with demi-water, the plates were developed with 100% methanolduring 30 minutes on a plate shaker. Absorbance was measured with amicroplate reader using a 550 nm filter with a 650 nm reference filter.Inhibition is measured in triplicates. Percentage of relative cellproliferation was determined by dividing the average absorbance from thetriplicate of a particular antibody concentration by the averageabsorbance from wells which had no antibody added, then times 100.

Effector Cell Isolation:

Peripheral white blood cells were isolated by a method slightly modifiedfrom that described in Repp, et al. (1991) Blood 78: 885-889. Briefly,heparin-anticoagulated blood was layered over a ficoll gradient. Aftercentrifugation, effector cells were harvested from the interphase andthe remaining erythrocytes were removed by hypotonic lysis. Cytospinpreparations were used to assess the purity of isolated cells which washigher than 95%. The viability of cells, determined by trypan blueexclusion, exceeded 95%.

ADCC Assays:

The capacity of fully human antibodies to lyse tumor cells was evaluatedin ⁵¹Chromium release assays (Valerius, et al. (1993) Blood, 82:931-939). Isolated human white blood cells were used as effector source.In brief, tumor targets were incubated with 100 μCi ⁵¹Cr for two hours.After a three times wash with culture medium, 5×10³ target cells wereadded to round-bottomed tissue culture plates containing 50 μl ofisolated effector cells and sensitizing MAb in different concentrationsand diluted in culture medium. The final volume was 200 μl and theeffector to target cell ratio (E:T) 80:1. The assays were incubatedovernight at 37° C. and stopped by centrifugation. The chromium releasewas measured in supernatants in triplicates. Percentage of cellularcytotoxity was calculated using the formula:

% Specific Lysis=Experimental cpm−Spontaneous cpm/Maximumcpm−Spontaneous cpm×100

with maximal ⁵¹Cr release determined by adding ZAP-oglobin® (10% finalconcentration) to target cells and basal release measured in the absenceof sensitizing antibodies and effector cells. Only very low levels ofantibody mediated, non-cellular cytotoxicity (without effector cells)was observed under these assay conditions (<5% specific lysis).

Affinity Measurements Using SPR Technology:

Binding affinity of anti EGFR antibodies was determined using BIAcore300 (Biacore, Upsula, Sweden). EGFR purified from A431 cells purchasedfrom Sigma was immobilized on a CMS chip according to the manufacturer'sinstructions. Measurements were done with antibody F(ab′) fragments atdifferent concentrations. Association and dissociation constants weredetermined using BIAevaluation software (version 3.1).

Mice and Tumor Models:

Nude Balb/c mice (NuNu) were purchased from Harlan (Horst, TheNetherlands). All experiments described were performed with female miceof eight to twelve weeks old. Mice were housed in the Transgenic MouseFacility of the Central Laboratory Animal Facility (Utrecht, TheNetherlands) and experiments were approved by the Utrecht Universityanimal ethics committee. When participating in an experiment, mice werechecked thrice a week for signs of toxicity and discomfort includinglevel of activity, skin abnormalities, diarrhea, and general appearance.A well-established subcutaneous (s.c.) tumor model was used. Briefly thehigh EGFR expressing A431 cells were inoculated, on the right side ofthe mouse, at a dose of 3×10⁶ cells. The tumors grow uniform and can beeasily measured by vernier calipers. The tumor volume is reported aslength×width×height (in mm³). The monoclonal antibodies were injectedintraperitoneally (i.p.) according to the study protocol. The tumorcells were tested for stable EGFR expression after in vivo passage byflow cytometry and immunohistochemistry. In order to determinepharmacokinetics, mice, with and without tumors, were injected i.p. with2F8 antibody. Weekly blood samples were taken via the tail vein beforeand for six weeks after the injection. The samples were analyzed byhuman IgG ELISA.

Statistical Analysis:

Group data are reported as mean±standard error of the mean (SEM).Differences between groups are analyzed by unpaired (or, whereappropriate, paired) Student's t-test. Levels of significance areindicated. Significance was accepted at the p<0.05 level.

Example 1 Generation of Cmu Targeted Mice for the Production ofAnti-EGFR Human Antibodies, Also Referred to as “HuMabs” Construction ofa CMD Targeting Vector

The plasmid pICEmu contains an EcoRI/XhoI fragment of the murine Igheavy chain locus, spanning the mu gene, that was obtained from a Balb/Cgenomic lambda phage library (Marcu et al. Cell 22: 187, 1980). Thisgenomic fragment was subcloned into the XhoI/EcoRI sites of the plasmidpICEMI9H (Marsh et al; Gene 32, 481-485, 1984). The heavy chainsequences included in pICEmu extend downstream of the EcoRI site locatedjust 3′ of the mu intronic enhancer, to the XhoI site locatedapproximately 1 kb downstream of the last transmembrane exon of the mugene; however, much of the mu switch repeat region has been deleted bypassage in E. coli. The targeting vector was constructed as follows. A1.3 kb HindIII/SmaI fragment was excised from pICEmu and subcloned intoHindIII/SmaI digested pBluescript (Stratagene, La Jolla, Calif.). ThispICEmu fragment extends from the HindIII site located approximately 1 kb5′ of Cmu1 to the SmaI site located within Cmu1. The resulting plasmidwas digested with SmaI/SpeI and the approximately 4 kb SmaI/XbaIfragment from pICEmu, extending from the SmaI site in Cmu1 3′ to theXbaI site located just downstream of the last Cmu exon, was inserted.The resulting plasmid, pTAR1, was linearized at the SmaI site, and a neoexpression cassette inserted. This cassette consists of the neo geneunder the transcriptional control of the mouse phosphoglycerate kinase(pgk) promoter (XbaI/TaqI fragment; Adra et al. (1987) Gene 60: 65-74)and containing the pgk polyadenylation site (PvuII/HindIII fragment;Boer et al. (1990) Biochemical Genetics 28: 299-308). This cassette wasobtained from the plasmid pKJ1 (described by Tybulewicz et al. (1991)Cell 65: 1153-1163) from which the neo cassette was excised as anEcoRI/HindIII fragment and subcloned into EcoRI/HindIII digestedpGEM-7Zf (+) to generate pGEM-7 (KJ1). The neo cassette was excised frompGEM-7 (KJ1) by EcoRI/SalI digestion, blunt ended and subcloned into theSmaI site of the plasmid pTAR1, in the opposite orientation of thegenomic Cmu sequences. The resulting plasmid was linearized with Not I,and a herpes simplex virus thymidine kinase (tk) cassette was insertedto allow for enrichment of ES clones bearing homologous recombinants, asdescribed by Mansour et al. (1988) Nature 336: 348-352. This cassetteconsists of the coding sequences of the tk gene bracketed by the mousepgk promoter and polyadenylation site, as described by Tybulewicz et al.(1991) Cell 65: 1153-1163. The resulting CMD targeting vector contains atotal of approximately 5.3 kb of homology to the heavy chain locus andis designed to generate a mutant mu gene into which has been inserted aneo expression cassette in the unique SmaI site of the first Cmu exon.The targeting vector was linearized with PvuI, which cuts within plasmidsequences, prior to electroporation into ES cells.

Generation and Analysis of Targeted ES Cells

AB-1 ES cells (McMahon, A. P. and Bradley, A., (1990) Cell 62:1073-1085) were grown on mitotically inactive SNL76/7 cell feeder layers(ibid.) essentially as described (Robertson, E. J. (1987) inTeratocarcinomas and Embryonic Stem Cells: a Practical Approach (E. J.Robertson, ed.) Oxford: IRL Press, p. 71-112). The linearized CMDtargeting vector was electroporated into AB-1 cells by the methodsdescribed Hasty et al. (Hasty, P. R. et al. (1991) Nature 350: 243-246).Electroporated cells were plated into 100 mm dishes at a density of1−2×10⁶ cells/dish. After 24 hours, G418 (200 micrograms/ml of activecomponent) and FIAU (5×10⁻⁷ M) were added to the medium, anddrug-resistant clones were allowed to develop over 8-9 days. Clones werepicked, trypsinized, divided into two portions, and further expanded.Half of the cells derived from each clone were then frozen and the otherhalf analyzed for homologous recombination between vector and targetsequences.

DNA analysis was carried out by Southern blot hybridization. DNA wasisolated from the clones as described Laird et al. (Laird, P. W. et al.,(1991) Nucleic Acids Res. 19: 4293). Isolated genomic DNA was digestedwith SpeI and probed with a 915 by SacI fragment, probe A, whichhybridizes to a sequence between the mu intronic enhancer and the muswitch region. Probe A detects a 9.9 kb SpeI fragment from the wild typelocus, and a diagnostic 7.6 kb band from a mu locus which hashomologously recombined with the CMD targeting vector (the neoexpression cassette contains a SpeI site). Of 1132 G418 and FIAUresistant clones screened by Southern blot analysis, 3 displayed the 7.6kb SpeI band indicative of homologous recombination at the mu locus.These 3 clones were further digested with the enzymes BglI, BstXI, andEcoRI to verify that the vector integrated homologously into the mugene. When hybridized with probe A, Southern blots of wild type DNAdigested with BglI, BstXI, or EcoRI produce fragments of 15.7, 7.3, and12.5 kb, respectively, whereas the presence of a targeted mu allele isindicated by fragments of 7.7, 6.6, and 14.3 kb, respectively. All 3positive clones detected by the SpeI digest showed the expected BglI,BstXI, and EcoRI restriction fragments diagnostic of insertion of theneo cassette into the Cmu1 exon.

Generation of Mice Bearing the Mutated Mu Gene

The three targeted ES clones, designated number 264, 272, and 408, werethawed and injected into C57BL/6J blastocysts as described by Bradley(Bradley, A. (1987) in Teratocarcinomas and Embryonic Stem Cells: aPractical Approach. (E. J. Robertson, ed.) Oxford: IRL Press, p.113-151). Injected blastocysts were transferred into the uteri ofpseudopregnant females to generate chimeric mice representing a mixtureof cells derived from the input ES cells and the host blastocyst. Theextent of ES cell contribution to the chimera can be visually estimatedby the amount of agouti coat coloration, derived from the ES cell line,on the black C57BL/6J background. Clones 272 and 408 produced only lowpercentage chimeras (i.e. low percentage of agouti pigmentation) butclone 264 produced high percentage male chimeras. These chimeras werebred with C57BL/6J females and agouti offspring were generated,indicative of germline transmission of the ES cell genome. Screening forthe targeted mu gene was carried out by Southern blot analysis of BglIdigested DNA from tail biopsies (as described above for analysis of EScell DNA). Approximately 50% of the agouti offspring showed ahybridizing BglI band of 7.7 kb in addition to the wild type band of15.7 kb, demonstrating a germline transmission of the targeted mu gene.

Analysis of Transgenic Mice for Functional Inactivation of Mu Gene

To determine whether the insertion of the neo cassette into Cmu1 hasinactivated the Ig heavy chain gene, a clone 264 chimera was bred with amouse homozygous for the JHD mutation, which inactivates heavy chainexpression as a result of deletion of the JH gene segments (Chen et al,(1993) Immunol. 5: 647-656). Four agouti offspring were generated. Serumwas obtained from these animals at the age of 1 month and assayed byELISA for the presence of murine IgM. Two of the four offspring werecompletely lacking IgM (see Table 2). Genotyping of the four animals bySouthern blot analysis of DNA from tail biopsies by BglI digestion andhybridization with probe A (see FIG. 1), and by StuI digestion andhybridization with a 475 bp EcoRI/StuI fragment (ibid.) demonstratedthat the animals which fail to express serum IgM are those in which oneallele of the heavy chain locus carries the JHD mutation, the otherallele the Cmu1 mutation. Mice heterozygous for the JHD mutation displaywild type levels of serum Ig. These data demonstrate that the Cmu1mutation inactivates expression of the mu gene.

TABLE 2 Serum IgM Mouse (micrograms/ml) Ig H chain genotype 42 <0.002CMD/JHD 43 196 +/JHD 44 <0.002 CMD/JHD 45 174 +/JHD 129 × BL6 F1 153 +/+JHD <0.002 JHD/JHDTable 2 shows the levels of serum IgM, detected by ELISA, for micecarrying both the CMD and JHD mutations (CMD/JHD), for mice heterozygousfor the JHD mutation (+/JHD), for wild type (129Sv x C57BL/6J)F1 mice(+/+), and for B cell deficient mice homozygous for the JHD mutation(JHD/JHD).

Example 2 Generation of HCO12 Transgenic Mice for the Production ofAnti-EGFR Human Antibodies The HCO12 Human Heavy Chain Transgene

The HCO12 transgene was generated by coinjection of the 80 kb insert ofpHC2 (Taylor et al., 1994, Int. Immunol., 6: 579-591) and the 25 kbinsert of pVx6. The plasmid pVx6 was constructed as described below.

An 8.5 kb HindIII/SalI DNA fragment, comprising the germline humanVH1-18 (DP-14) gene together with approximately 2.5 kb of 5′ flanking,and 5 kb of 3′ flanking genomic sequence was subcloned into the plasmidvector pSP72 (Promega, Madison, Wis.) to generate the plasmid p343.7.16.A 7 kb BamHI/HindIII DNA fragment, comprising the germline human VH5-51(DP-73) gene together with approximately 5 kb of 5′ flanking and 1 kb of3′ flanking genomic sequence, was cloned into the pBR322 based plasmidcloning vector pGP1f (Taylor et al. 1992, Nucleic Acids Res. 20:6287-6295), to generate the plasmid p251f. A new cloning vector derivedfrom pGP1f, pGP1k, was digested with EcoRV/BamHI, and ligated to a 10 kbEcoRV/BamHI DNA fragment, comprising the germline human VH3-23 (DP47)gene together with approximately 4 kb of 5′ flanking and 5 kb of 3′flanking genomic sequence. The resulting plasmid, p112.2RR.7, wasdigested with BamHI/SalI and ligated with the 7 kb purified BamHI/SalIinsert of p251f. The resulting plasmid, pVx4, was digested with XhoI andligated with the 8.5 kb XhoI/SalI insert of p343.7.16.

A clone was obtained with the VH1-18 gene in the same orientation as theother two V genes. This clone, designated pVx6, was then digested withNotI and the purified 26 kb insert coinjected—together with the purified80 kb NotI insert of pHC2 at a 1:1 molar ratio—into the pronuclei ofone-half day (C57BL/6J x DBA/2J)F2 embryos as described by Hogan et al.(B. Hogan et al., Manipulating the Mouse Embryo, A Laboratory Manual,2^(nd) edition, 1994, Cold Spring Harbor Laboratory Press, PlainviewN.Y.). Three independent lines of transgenic mice comprising sequencesfrom both Vx6 and HC2 were established from mice that developed from theinjected embryos. These lines are designated (HCO12)14881, (HCO12)15083,and (HCO12)15087. Each of the three lines were then bred with micecomprising the CMD mutation described in Example 1, the JKD mutation(Chen et al. 1993, EMBO J. 12: 811-820), and the (KCoS)9272 transgene(Fishwild et al. 1996, Nature Biotechnology 14: 845-851). The resultingmice express human immunoglobulin heavy and kappa light chain transgenesin a background homozygous for disruption of the endogenous mouse heavyand kappa light chain loci.

Example 3 Production of Human Monoclonal Antibodies Against EGFR

Two different strains of mice were used to generate EGFR reactive humanmonoclonal antibodies. Strain ((CMD)++; (JKD)++; (HCo7)11952+/++;(KCoS)9272+/++) (referred to herein as “HCO7 mice”, and strain ((CMD)++;(JKD)++; (HCo12)15087+/++; (KCoS)9272+/++) (referred to herein as “HCO12mice”). Each of these strains are homozygous for disruptions of theendogenous heavy chain (CMD) and kappa light chain (JKD) loci. Bothstrains also comprise a human kappa light chain transgene (HCo7), withindividual animals either hemizygous or homozygous for insertion #11952.The two strains differ in the human heavy chain transgene used. Micewere hemizygous or homozygous for either the HCo7 or the HCo12transgene. The CMD mutation is described above in Example 1. Thegeneration of (HCo12)15087 mice is described in Example 2. The JKDmutation (Chen et al. 1993, EMBO J. 12: 811-820) and the (KCoS)9272(Fishwild et al. 1996, Nature Biotechnology 14: 845-851) and (HCo7)11952mice, are described in U.S. Pat. Nos. 5,770,429 and 5,545,806 (Lonberg &Kay, Jun. 23, 1998).

The immunization schedule used is listed in Table 3 below. Mice wereimmunized twice with A 431 cells followed by soluble antigen in RibiAdjuvant. The EGFR specific serum titer was determined by ELISA afterthe third immunization. Three different immunizations were done for thefinal boosts before the fusion. These included two or three sequentialintravenous (iv) boosts via the tail vein with 10 μg of antigen in 50 μlPBS or two sequential intraperitoneal (i.p.) boosts with 25 μg solubleEGFR in Ribi adjuvant (see Table 3). The three mice that were used inthe fusion were part of a larger cohort of mice that included both HCo7and HCo12 genotypes.

TABLE 3 Immunization Schedule EGFR ELISA in Ribi ELISA EGFR A431 cellsA431 cells Titer ip Titer Fusion in RIBI ip Fusion Mouse Day 1 Day 20Day 30 Day 33 Day 43 Day 46 Day 50 Day 53 20241 2 × 10⁶ 1 × 10⁷ 0 25 μg4050 25 μg Ribi 2 × 25 μg*** 20242 2 × 10⁶ 1 × 10⁷ 0 25 μg 4050 25 μg 2iv × 10 μg* 20243 2 × 10⁶ 1 × 10⁷ 450 25 μg 12150 3 iv × 10 μg* *EGFR inPBS (10 μg) iv on days −4, −3, and −2 **EGFR in PBS (10 μg) iv on day−4, and −3 ***EGFR in Ribi (25 μg) i.p. on day −4 and −3

Immunization strategy used for the first two injections, 2−10×10⁶ liveA431 cells i.p., resulted in poor anti-EGFR titers (see Table 2).However, when these mice were given a third immunization with 25μg/mouse of soluble EGFR in Ribi adjuvant, serum titers increased morethan 30 fold. These results clearly demonstrate that cells expressing alarge amount of EGFR on the cell surface are very effective atinitiating a primary immune response that then was greatly enhanced withonly one dose of purified antigen in adjuvant.

The final boost before fusion for mouse 20243 was done as i.v. tail veinboosts with 10 μg soluble EGFR in PBS on days −4, −3, and −2. The TritonX-100 in the soluble EGFR caused an irritation to the tail of the mouse.Therefore, to reduce the possibility of irritation, mouse 20242 receivedonly two i.v. vein boosts with soluble EGFR on days −4 and −3, and mouse20241 received two i.p. immunizations on days −4 and −3 with 25 μg EGFRin Ribi adjuvant. The three fusions resulted in 46 human γ, κ-antigenpositive hybridomas (see Table 4). Mouse 20241 alone, which received thei.p. boosts with adjuvant, produced 35 antigen specific Human GammaKappa antibodies.

TABLE 4 γ/κ+ γ1κ+ γ3κ+ Mouse γκ+ EGFR+ EGFR+ EGFR+ 20243 120 14 13 120242 35 2 2 0 20241 * 30 28 2

Example 4 Hybridoma Preparation

The P3 X63 ag8.653 myeloma cell line (ATCC CRL 1580, lot F-15183) wasused for the fusions. The original ATCC vial was thawed and expanded inculture. A seed stock of frozen vials was prepared from this expansion.A fresh vial of cells was thawed one to two weeks before the fusions.

High Glucose DMEM (Mediatech, Cellgro #10013) containing 10% FBS,Pennicillin-Streptomycin (Sigma, P-7539), and 5.5×10⁻⁵M2-mercaptoethanol (GibcoBRL, 21985-023) was used to culture A431 cellsand myeloma cells. Additional media supplements were added to theHybridoma growth media, which included: 3% Origin-Hybridoma CloningFactor (Igen, 21001), OPI supplement (Sigma, 0-5003), 1.1×10⁻³ M Oxaloacetic acid, 4.5×10⁴ M sodium Pyruvate, and 24 international units/Lbovine Insulin, HAT (Sigma, H 0262) 1.0×10⁴ M Hypoxanthine, 4.0×10⁻⁷ MAminopterin, 1.6×10⁻⁵ M Thymidine, or HT (Sigma, H0137) 1.0×10⁴ MHypoxanthine, 1.6×10⁻⁵ M Thymidine.

Characterized Fetal bovine serum (SH30071 lot #s AJE10321 and AGH6843)was obtained from Hyclone, Logan, Utah. Serum Free medium containedDMEM, antibiotics and 2-mercaptoethanol only.

Spleens from all three mice were normal in size and yielded from 2×10⁷to 1×10⁸ splenocytes. The splenocytes were fused.

The initial ELISA screen for human IgG κ antibodies was performed 7-10days post fusion. Human IgG, κ positive wells were screened on solubleEGFR coated ELISA plates. Antigen positive hybridomas were transferredto 24 well plates and eventually to tissue culture flasks. EGFR specifichybridomas were subcloned by limiting dilution to assure monoclonality.Antigen positive hybridomas were preserved at several stages in thedevelopment process by freezing cells in DMEM 10% FBS plus 10% DMSO(Sigma, D2650) or in Origen Freeze Medium (Igen, #210002). Cells werestored at −80° C. or in LN₂.

Initial EGFR specific hybridomas were subsequently evaluated for epitopespecificity and their ability to block the binding of EGF to the EGFRreceptor. Mouse monoclonal anti-EGFR antibodies 225 and 528 havepreviously been shown to bind to EGFR, block binding of EGF to EGFR andto be anti-cancer immunotherapeutic agents in animal and human studies.Therefore these antibodies were used, in addition to a non-blockingantibody, in a competitive ELISA format to identify human antibodiesthat have immunotherapeutic characteristics.

Example 5 Binding Affinity

Binding affinity for hybridoma 2F8 was determined using BIAcore 3000(Biacore, Upsula, Sweden). EGFR purified from A431 cells purchased fromSigma was immobilized on a CMS chip according to the manufacturer'sinstructions. Antibody 2F8 had an equilibrium association constant(K_(A)) of 5.47 (±0.52)×10⁸ M⁻¹.

Example 6 Competitive ELISA Assays

Competitive ELISA assays were used as the initial qualifying assay assoon as antigen positive hybridomas were established in 24 well plates.In general, strong competition (80-100%) indicates that an antibodybinds to the same epitope or to a region of the antigen in closeproximity to the competing antibody. Weaker competition of less than 50%indicates that the antibody and its competitor bind to regions of theantigen not in close proximity. Initial assays were done withsupernatants from uncloned hybridomas many of which contained more thanone hybridoma per well. Later assays were done with subclones of theoriginal wells. FIGS. 1 and 2 show (the data in FIGS. 1 and 2 arearranged based on degree of competition with MAb 225) that even withcrude cell culture supernatants, antibodies can be identified that bindto similar or identical epitopes as the 225 and 528 antibodies. Alsoevident in this experiment is the different distribution in competitivebinding patterns of antibodies derived from mouse 20241 or from mouse20242 and 20243. For example, the first seven antibodies from the #20241mouse (FIG. 1) compete strongly with both MAb 225 and 528. The remainderof the antibodies from 20241 competed moderately or weakly with the 225and 528 antibodies. Five antibodies (1H6, 2F8, 1A8, 5C5, and 8E1) fromthe 20242 and 20243 mice showed strong competition with antibody 225 andno competition or weak competition from MAb 528 (FIG. 2). Antibodies2F6, 8A12, 5F12, 6B3, and 6D9 from mouse 20242 and 20243 competed withboth MAb 225 and 528, although the competition was stronger against the225 antibody. Other antibodies from these mice did not compete or wereweakly competitive with the commercial MAbs.

These initial competitive ELISA results were verified with purifiedantibodies produced by sub-cloned cells. FIGS. 3 and 4 show thatantibodies 5F12 and 6B3 compete strongly with both MAb 225 and 528 andalso demonstrate reciprocal competition with each other. This dataindicate that these antibodies bind to the same epitope or to a regionof the EGFR molecule in close proximity to the 225 or 528 binding site.Antibody 2F8 competes moderately with MAb 225 and does not significantlycompete with antibody 528 (FIGS. 3 and 4). However, antibody 2F8, 6B3and 5F12 show strong cross competition. This data suggests that antibody2F8 is binding to a separate epitope from the 225 and 528 antibodies andbinds to a region of the EGFR receptor that is adjacent to or overlapswith the epitope to which HuMabs 6B3 and 5F12 bind. Antibodies 2A2 and6E9 do not compete with either MAb and bind to EGFR epitopes unrelatedto the binding sites of the 225 and 528 MAbs (FIGS. 3 and 4).

Example 7 EGF/EGFR Blocking Assays

Antigen positive subclones were further evaluated in EGF/EGFR blockingassays. These assays included subclones of antibodies that competestrongly with MAb 225 and/or 528, as well as, antibodies that are weakor non competitive with 225 or 528. Several antibodies were expanded inculture medium and purified by protein A chromatography. FIGS. 5 and 6show that antibodies 2F8, 5F12, and 6B3, which are moderate to strongcompetitors of the 225 antibody in ELISA, are strong blockers of EGFbinding to EGFR. This is evident in assays done in ELISA format or byFACS on human A431 epidermoid cancer cells. In both assays, the humanantibodies were as good as or better than MAb 225. Antibodies 2F8, 5F12,6B3, and 6E9 also have similar binding characteristics on the surface ofA431 cells (FIG. 7).

The in vitro EGF/EGFR blocking and ELISA competition studiesdemonstrated that the 2F8, 5F12, and 6B3 antibodies have similarproperties to other anti-EGFR murine and human antibodies that have beenshown to be immunotherapeutic agents (Sato, et al. (1983) Mol. Biol.Med. 511-529; Gill, et al. (1984) J. of Biol. Chem. 259(12):7755-7760).The 2F8 antibody was equivalent to or better than the 6B3 and 5F12antibodies overall in the various evaluations.

Example 8 Inhibition of EGF/TGF-α Binding to the EGF Receptor UsingHuman Monoclonal Antibodies to the EGF Receptor

Inhibition studies were performed on A431 cells using flow cytometry,ELISA, and inhibition of ligand-induced autophosphorylation. Murine MAbs225 or 525 were used as positive controls. An irrelevant human IgGisotype control, was used as an isotype control. A single humanantibody, 2F8, was chosen for all the further studies. This antibody isalso referred to herein as “Humax-EGFR™”. FIG. 8 shows the EGF blockingcapacity of 2F8 in a concentration dependent manner. 2F8 and m225 blockto the same extent while the blocking capacity of EGF is less.

FIG. 9 further shows the blocking capacity of 2F8 in that it efficientlyinhibits the binding of EGF and TGF-α to A431 cells (cells derived froman ovarian epidermoid carcinoma and express in excess of 1×10⁶ EGFRmolecules on their cell surface). Inhibition of 2F8-binding to A431cells was determined using flow cytometer analysis. Cells werepre-incubated with either 5 (open bars) or 50 μg/ml (closed bars) ligandbefore adding 2F8. Binding of antibody without ligand (PBS group) wasdesignated as 100%. These results indicate that 2F8 binds close to, orat the same site, on EGFR as the ligands.

Example 9 Inhibition of Tumor Cell Activation Using Human MonoclonalAntibodies to the EGF Receptor

To evaluate the ability of 2F8 to inhibit tumor cell activation, theeffect of 2F8 on EGF-triggered cellular responses, such as activation ofthe intrinsic tyrosine kinase activity and concomitant cellproliferation, was examined. One of the first events after EGF or TGF-αbinding to the EGFR is the induction of autophosphorylation of thereceptor. Incubation of EGF with A431 cells results in tyrosinephosphorylation of the EGFR (M_(r) 170,000) (FIG. 10A). While 2F8 didnot activate the receptor kinase activity by itself, the antibodyblocked EGF-triggered EGFR tyrosine phosphorylation in a dose-dependentmanner with a complete inhibition at a concentration of 16.6 nM(antibody:EGF molar ratio, 20:1, FIG. 10A). Cells were treated withantibody and TGF-α showed that tyrosine phosphorylation was fullyblocked by 2F8 at a concentration of 66 nM (antibody: TGF-α molar ratio,7, 3:1, FIG. 10B).

Engagement of EGF/TGF-α with the receptor results in cell activationwhich is reflected in cell proliferation. Therefore the inhibitoryeffect of 2F8 on growth of tumor cells (A431, MDA-MD-468 and HN5 cells)was evaluated. The experiments were carried out in the absence ofexogenous EGF. Mouse antibodies were used as a comparison. Humax-EGFRinhibited the growth of A431 cells in a concentration dependent mannerwith a maximal inhibition of 50%, a level similar to that obtained withmouse antibody 225 (FIG. 14). The control antibody had no effect on thecell proliferation (FIG. 14). Growth inhibition was also obtained withtwo other cell lines at similar levels (HN5 and MDA-MB468, panels B andC). As no exogenous EGF was added to the culture, these results indicatethe ability of 2F8 to block autocrine stimulation and thus to inhibitautocrine EGF/TGF-α induced tumor cell activation.

Example 10 Human Monoclonal Antibodies to the EGF Receptor Induce ADCC

ADCC is a potent immune effector mechanism triggered by the recognitionof tumor cells by antibodies. To evaluate the ability of human PMN cellsto kill A431 cells in the presence of 2F8, A431 cells were loaded with⁵¹Cr and subsequently incubated with antibody and effector cells (PMN)overnight. After incubation, chromium release was measured. As shown inFIG. 14, 2F8 is capable of inducing ADCC against A431 cells using humanPMN. 2F8 is capable of mediating PMN-induced lysis of 45% of the A431target cells, which is higher then observed with the MAb 425 (FIG. 14).

Importantly, while capable of recruiting immune effector cells andinducing ADCC, 2F8 is unable to induce complement-mediated lysis oftumor cells.

Example 11 Human Monoclonal Antibodies to the EGF Receptor Prevent TumorFormation

To show the ability of HuMab 2F8 to prevent tumor formation in anathymic murine model, groups of six (6) mice were injectedsubcutaneously in the flank with 3×10⁶ tumor cells in 200 μl PBS at dayzero (0). Subsequently, mice were injected i.p. on days 1 (75 μg/200μl), 3 (25 μg/200 μl), and 5 (25 μg/200 μl) (arrows) with either HuMab2F8 (closed squares) i.p. of human IgG1-κ MAb as a control (opencircles) (FIG. 14). The data are presented as mean tumor volume+SEM, andare representative of 3 individual experiments, yielding similarresults.

Eradication of established A431 tumor xenografts by HuMab 2F8 incomparison to m225 is shown in FIG. 14. Mice were injectedsubcutaneously in the flank with 3×10⁶ tumor cells in 200 μl PBS on dayzero (0). At day 10, mice were randomly allocated to treatment groupsand treated on days 12 (75 μg/200 μl), 14 (25 μg/200 μl), and 16 (25μg/200 μl) (arrows) with HuMab 2F8 (closed squares, 2F8 short-term) orwith murine anti-EGFR MAb m225 (closed triangles, m225 short-term).Furthermore, groups were included receiving 75 μg/200 μl HuMab 2F8 orm225 on day 12, continued by 25 μg/200 μl HuMab 2F8 or m225 on days 14,16, 19, 22, 26, 29, 33, 36, and 40 (open squares, 2F8 long-term; opentriangles, m225 long-term). The data are presented as mean tumorvolume+SEM, and are representative of 3 individual experiments, yieldingsimilar results. Black arrows indicate treatment days for the short-termtreatment, open arrows indicate treatment days for the long-termtreatment.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

All patents, pending patent applications and other publications citedherein are hereby incorporated by reference in their entirety. Anycombination of the embodiments disclosed in the dependent claims arecontemplated to be within the scope of the invention.

1-95. (canceled)
 96. An isolated human monoclonal antibody which bindsto human EGFR comprising heavy and light chain variable regions, whereinthe heavy chain variable region is encoded by the nucleic acid set forthin SEQ ID NO: 1, or the light chain variable region is encoded by thenucleic acid sequence set forth in SEQ ID NO:
 3. 97. An isolated humanmonoclonal antibody which binds to human EGFR comprising heavy and lightchain variable regions, wherein the heavy chain variable regioncomprises the CDR3 sequence set forth in SEQ ID NO: 7, and the lightchain variable region comprises the CDR3 sequence set forth in SEQ IDNO:
 10. 98. The antibody of claim 96, wherein the antibody is an IgG1antibody.
 99. The antibody of claim 96, wherein the antibody binds tohuman EGFR with an equilibrium association constant (K_(A)) of at leastabout 10⁸M⁻¹.
 100. The antibody of claim 96, wherein the antibody: (a)inhibits EGFR ligand binding to EGFR; (b) has a binding equilibriumassociation constant (K_(A)) to human EGFR of at least about 10⁷M⁻¹; and(c) inhibits growth of a cell expressing EGFR.
 101. The antibody ofclaim 100, wherein the cell is a tumor cell selected from the groupconsisting of a bladder cell, a breast cell, a colon cell, a kidneycell, an ovarian cell, a prostate cell, a renal cell, a squamous cell,and a non-small lung cell.
 102. The antibody of claim 101 wherein thecell is selected from the group consisting of a synovial fibroblast celland a keratinocyte.
 103. The antibody of claim 101 which binds to cellsexpressing EGFR and induces lysis (ADCC) of the cells in the presence ofhuman effector cells and/or does not induce complement-mediated lysis ofthe cells in vivo.
 104. The antibody of claim 96, which binds to EGFRand inhibits EGF- or TGF-α-induced autophosphorylation of EGFR.
 105. Anantigen-binding portion of the antibody of claim 96 comprising a Fabfragment or a single chain antibody.
 106. The antibody of claim 96produced by a hybridoma which includes a B cell obtained from atransgenic non-human animal having a genome comprising a human heavychain transgene and a human light chain transgene fused to animmortalized cell.
 107. The antibody of claim 96, wherein the antibodyhas a dissociation constant (K_(D)) from EGFR of about 10⁻³ s⁻¹ or less.108. A bispecific antibody comprising a first antigen-binding region anda second antigen-binding region, wherein the first antigen-bindingregion binds the same epitope on EGFR as the antibody of claim 96, andwherein the second antigen-binding region binds a humanantigen-presenting cell (APC).
 109. A bispecific antibody comprising afirst antigen-binding region and a second antigen-binding region,wherein the first antigen-binding region binds the same epitope on EGFRas the antibody of claim 96, and wherein the second antigen-bindingregion binds a human Fc receptor.
 110. The bispecific antibody of claim109 wherein the Fc receptor is a human FcγRI or a human Fcα receptor.111. The bispecific antibody of claim 109 which binds to the Fc receptorat a site which is distinct from the immunoglobulin binding site of thereceptor.
 112. A composition comprising the antibody of claim 96 and acarrier.
 113. A composition comprising the antibody of claim 96 and achemotherapeutic agent.
 114. An immunotoxin comprising the antibody ofclaim 96 linked to a cytotoxic agent.
 115. A method of detecting thepresence of EGFR antigen, or a cell expressing EGFR, in a samplecomprising: contacting the sample with an antibody comprising heavy andlight chain variable region sequences which comprise the amino acidsequences as set forth in SEQ ID NO: 2 and SEQ ID NO: 4, respectively,under conditions that allow for formation for a complex between theantibody, or portion thereof, and EGFR; and detecting the formation of acomplex.